Microrna profiling for diagnosis of dysplastic nevi and melanoma

ABSTRACT

Provided herein are methods for miRNA profiling for the diagnosis, prognosis, and management of melanoma and differentiation of melanoma from nevi.

FIELD OF THE INVENTION

The invention generally relates to miRNA profiling for the diagnosis, prognosis, and management of melanoma and differentiation of melanoma from nevi.

BACKGROUND OF THE INVENTION

The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.

Skin cancer is the most common of all cancers, afflicting more than a million Americans each year, a number that is rising rapidly. It is also the easiest to cure, if diagnosed and treated early. If allowed to progress to the point where it spreads to other sites (metastasizes), the prognosis (forecast) is very poor. More than 8,000 melanoma deaths now occur per year.

Melanoma most often appears as an asymmetrical, irregularly bordered, multicolored or tan/brown spot or growth that continues to increase in size over time. It may begin as a flat spot and become more elevated. In rare instances, it may not be pigmented.

Dysplastic nevi (atypical moles) are unusual or benign moles that may resemble melanoma. People who have them are at increased risk of developing single or multiple melanomas. The higher the number of these moles someone has, the higher the risk; those who have 10 or more have 12 times the risk of developing melanoma compared to the general population. Dysplastic nevi are found significantly more often in melanoma patients than in the general population.

Melanoma is distinguished from nevi, other forms of cancer, and normal skin on the basis of clinical presentation and histopathological examination of a skin biopsy, usually a formalin fixed, paraffin embedded (FFPE) sample. Considerable expertise is required to reliably distinguish between nevi and melanoma.

This application describes novel microRNA biomarkers with microRNA array and RT-PCR to better characterize dysplastic nevi, malignant melanoma and metastatic melanoma, miRNA can therefore serve as an adjunct to histopathology for correct classification of melanoma, nevi and other conditions, especially where there is doubt as to the diagnosis.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that melanoma can be distinguished from nevi by measuring changes in the levels of as little as two miRNAs.

In one aspect, the invention provides a method for differentially diagnosing melanoma from nevi, by (a) measuring the level of two or more miRNAs selected from the group consisting of miR-132, miR-150, miR-339-5p, miR-15b, miR-342-3p, miR-572, miR-155, miR-425, miR-1202, miR-1268, HBII-382_s, miR-1225-5p, miR-30c, miR-106b-star, miR-125a-5p, mgU6-53B, miR-25, miR-149-star, miR-939, miR-92b-star, miR-500-star, miR-22, HBII-142_x, miR-181b, HBII-142, U38B, miR-663, miR-1224-5p, miR-23a, HBII-85-6_x, miR-1207-5p, miR-1301, miR-1228-star, miR-345, miR-30a-star, ENSG00000199411, ENSG00000202327, miR-92a, miR-127-3p, HBII-85-26, miR-1308, miR-31, miR-921, miR-146b-5p, miR-768-3p, miR-708, miR-139-5p, ACA24_x, miR-501-3p, miR-502-3p, miR-923, and miR-191; and (h) diagnosing the skin sample as containing melanoma when a difference in the level of the two or more miRNAs compared to a reference level indicates melanoma in the sample. The level of two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more, miRNAs are measured.

In specific embodiments, the miRNAs to be measured include the following combinations (i) miR-150 and miR-149-star; (ii) miR-150, miR-149-star and miR-1308; (iii) miR-150, miR-149-star, miR-1308, and miR-191; (iv) miR-150, miR-149-star, miR-1308, miR-191, and miR-1228-star; (v) miR-150, miR-149-star, miR-1308, miR-191, miR-1228-star, and ENSG00000199411; (vi) miR-150, miR-149-star, miR-1308, miR-191, miR-1228-star, ENSG00000199411, and miR-1268; (vii) miR-150, miR-149-star, miR-1308, miR-191, miR-1228-star, ENSG00000199411, miR-1268, and miR-923; (viii) miR-150, miR-149-star, miR-1308, miR-191, miR-1228-star, ENSG00000199411, miR-1268, miR-923, and miR-23a; (ix) miR-150, miR-149-star, miR-1308, miR-191, miR-1228-star, ENSG00000199411, miR-1268, miR-923, miR-23a, and miR-132; (x) miR-150, miR-149-star, miR-1308, miR-191., miR-1228-star, ENSG00000199411, miR-1268, miR-923, miR-23a, miR-132, and miR-1207.5p; (xi) miR-150, miR-149-star, miR-1308, miR-191, miR-1228-star, ENSG00000199411, miR-1268, miR-923, miR-23a, miR-132, miR-1207.5p, and miR-342.3p; (xii) miR-150, miR-149-star, miR-1308, miR-191, miR-1228-star, ENSG00000199411, miR-1268, miR-923, miR-23a, miR-132, miR-1207.5p, miR-342.3p, and U38B; (xiii) miR-150, miR-149-star, miR-1308, miR-191, miR-1228-star, ENSG00000199411, miR-1268, miR-923, miR-23a, miR-132, miR-1207.5p, miR-342.3p, U38B, and miR-155.

In specific embodiments, melanoma is diagnosed in the skin sample by alterations in the level of an miRNA compared to a reference level, with the following alterations observed in at least two miRNA selected from the group consisting of: (i) miR-150 increase; (ii) miR-149-star decrease; (iii) miR-1308 decrease; (iv) miR-191 increase; (v) miR-1228-star decrease; (vi) ENSG00000199411_s decrease; (vii) miR-1268 decrease; (viii) miR-923 decrease; (ix) miR-23a increase; (x) miR-132 increase; (xi) miR-1207.5p decrease; (xii) miR-342.3p increase; (xiii) U38B decrease; and (xiv) miR-155 increase.

In one aspect, the invention provides a method for differentially diagnosing melanoma from nevi, by (a) measuring the level of two or more miRNAs selected from the group consisting of: miR-1268, miR-1228-star, miR-92b-star, miR-155, miR-345, miR-425, miR-132, miR-1207-5p, miR-1301, miR-663, miR-339-5p, miR-149-star, miR-150, miR-18a, miR-103, miR-191, miR-296-3p, miR-31, miR-107*, miR-93*, miR-1275*, miR-181B*, miR-921*, miR-1225-5p, miR-1202, and miR-342-3p and (b) diagnosing the skin sample as containing melanoma when a difference in the level of the two or more miRNAs compared to a reference level indicates melanoma in the sample.

In specific embodiments, melanoma is diagnosed in the skin sample by alterations in the level of an miRNA compared to a reference level, with the following alterations observed in at least two miRNA selected from the group consisting of: miR-1268 decrease, miR-1228-star decrease, miR-92b-star decrease, miR-155 increase, miR-345 increase, miR-425 increase, miR-132 increase, miR-1207-5p decrease, miR-1301 increase, miR-663 decrease, miR-339-5p increase, miR-149-star decrease, miR-150 increase, miR-18a increase, miR-103 increase, miR-191 increase, miR-296-3p decrease, miR-31 increase, miR-107* increase, miR-93* increase, miR-1275* decrease, miR-181B* increase, miR-921* decrease, miR-1225-5p increase, miR-1202 decrease, and miR-342-3p increase.

The method of the invention may further include internal controls, such as measuring the level of an miRNA selected from miR-27b, miR-195, miR-199b-3p, and miR-199a-3p.

In yet further embodiments, the level of two or more miRNAs are used to distinguish melanoma from normal skin, and nevi from normal skin. Additional miRNA levels may be assayed for this purpose.

The level of miRNA in the sample can be determined by microarray and/or quantitative real-time PCR. The method of the invention may be performed on a fresh skin sample, on a fixed and/or paraffin-embedded sample. In one embodiment the skin sample is formalin-fixed and paraffin-embedded.

The method of the invention may further comprise other steps in the diagnosis of melanoma, and the differentiation between nevi and melanoma, including histopathological assessment, and clinical assessment. In related embodiments, the clinical and/or histopathological evaluations may be converted into a score that can be combined with a score derived from miRNA levels, resulting in a diagnostic score that reflects the likelihood of melanoma.

The method of the invention may further include a step of isolating nucleic acids from the sample. An additional step may include amplification of the nucleic acid.

In further embodiments, the invention comprise a kit. In one embodiment, a kit for differentially diagnosing between melanoma and nevus in a skin sample comprises primers for the amplification of at least two miRNA selected from the group consisting of: miR-150, miR-149-star, miR-1308; miR-191.; miR-1228-star; ENSG00000199411_s; miR-1268; miR-923; miR-23a; miR-132; miR-1207.5p; miR-342.3p; U38B; and miR-155.

In another embodiment, the kit comprises primers for the amplification of at least two miRNAs selected from the group consisting of: miR-1268, miR-8-star, miR-92b-star, miR-155, miR-345, miR-425, miR-132, miR-1207-5p, miR-1301, miR-663, miR-339-5p, miR-149-star, miR-150, miR-18a, miR-103, miR-191, miR-296-3p, miR-31, miR-107*, miR-93*, miR-1275*, miR-181B*, miR-921*, miR-1225-5p, miR-1202, and miR-342-3p.

The kit may also primers for amplification of controls, such as miR-27b, miR-195, miR-199b-3p, and miR-199a-3p.

The kit may also include suitable buffers, reagents for isolating nucleic acid, and instructions for use Kits may also include a microarray for measuring miRNA levels.

BRIEF DESCRIPTION OF THE FIGURE

FIGS. 1-10 show, respectively, the best 2-10 miRNA combinations for differentiating melanoma (MM) from normal skin (NS), and the relevant error rates and AUC, as determined by different statistical algorithms.

FIGS. 11-23 show, respectively, the best 2-14 miRNA combinations for differentiating melanoma (MM) from nevi (NV), and the relevant error rates and AUC as determined by different statistical algorithms.

FIG. 24 shows the log 2 signal for three miRNA: miR-150 (miR-150), miR-149-star (miR-149-star), and hsa-miR-1308 (miR-1308) across 137 samples. Samples 1-19 are normal skin (NS), 20-57 nevi (NV), 58-115 melanoma (MM) and 116-137 metastatic melanoma (Mets). As can be appreciated, miR-150, miR-149-star and miR-1308 distinguish normal skin and nevi from melanoma and metastatic.

FIG. 25 shows an example of error rate and AUC from 9 programs analyzing the ability of groups of miRNA analytes to differentiate between melanoma and nevi.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have discovered that the levels of miRNAs in skin samples is a powerful tool to differentiate melanoma from non-tumorous nevi and, thereby, replace or supplement traditional clinical and histological methods of diagnosis.

Definitions

The present technology is described herein using several definitions, as set forth throughout the specification. As used herein, unless otherwise stated, the singular forms “a,” “an,” and “the” include plural reference. Thus, for example, a reference to “a nucleic acid” is a reference to one or more nucleic acids.

As used herein, “about” will be understood by persons of ordinary skill in the art and will vary to some extent depending upon the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art, given the context in which it is used, the term “about” in reference to quantitative measurements or values will mean up to plus or minus 10% of the enumerated value.

The term “amplification” or “amplify” as used herein means one or more methods known in the art for copying a target nucleic acid, thereby increasing the number of copies of a selected nucleic acid sequence. Amplification may be exponential or linear. A target nucleic acid may be either DNA or RNA. The sequences amplified in this manner form an “amplicon.” While the exemplary methods described hereinafter relate to amplification using the polymerase chain reaction (“PCR”), numerous other methods are known in the art for amplification of nucleic acids (e.g., isothermal methods, rolling circle methods, etc.). The skilled artisan will understand that these other methods may be used either in place of, or together with, PCR methods. See, e.g., Saiki, “Amplification of Genomic DNA” in PCR Protocols, Innis et al., Eds., Academic Press, San Diego, Calif. 1990, pp. 13-20; Wharam et al., Nucleic Acids Res., 2001, 29(11):E54-E54; Hafner et al., Biotechniques 2001, 30(4):852-6, 858, 860; Zhong et al., Biotechniques, 2001, 30(4):852-6, 858, 860.

The terms “determining,” “measuring,” “assessing,” and “assaying” are used interchangeably and include both quantitative and qualitative measurement, and include determining if a characteristic, trait, or feature is present or not. Assessing may be relative or absolute. “Assessing the presence of” includes determining the amount of something present, as well as determining whether it is present or absent.

The term “clinical factors” as used herein, refers to any data that a medical practitioner may consider in determining a diagnosis of melanoma. Such factors include, but are not limited to, the patient's medical history, age, gender, skin color, a physical examination of the patient, and histopathology.

The term “complement” used herein means the complementary sequence to a nucleic acid according to standard Watson/Crick base pairing rules. A complement sequence can also be a sequence of RNA complementary to the DNA sequence or its complement sequence, and can also be a cDNA. The term “substantially complementary” as used herein means that two sequences hybridize under stringent hybridization conditions. The skilled artisan will understand that substantially complementary sequences need not hybridize along their entire length. In particular, substantially complementary sequences comprise a contiguous sequence of bases that do not hybridize to a target or marker sequence, positioned 3′ or 5′ to a contiguous sequence of bases that hybridize under stringent hybridization conditions to a target or marker sequence.

As used herein, the term “diagnosis” means detecting melanoma or the presence of melanoma cells. Usually, a diagnosis of a disease or disorder is based on the evaluation of one or more factors and/or symptoms that are indicative of the disease. That is, a diagnosis can be made based on the presence, absence or amount of a factor which is indicative of presence or absence of the disease or condition. Each factor or symptom that is considered to be indicative for the diagnosis of a particular disease does not need be exclusively related to the particular disease; i.e. there may be differential diagnoses that can be inferred from a diagnostic factor or symptom. Likewise, there may be instances where a factor or symptom that is indicative of a particular disease is present in an individual that does not have the particular disease. The term “diagnosis” also encompasses determining the therapeutic effect of a drug therapy, or predicting the pattern of response to a drug therapy. The diagnostic methods may be used independently, or in combination with other diagnosing and/or staging methods known in the medical art for a particular disease or disorder, particularly melanoma.

As used herein, the phrase “difference of the level” refers to differences in the quantity of a particular marker, such as a nucleic acid or a protein, in a sample as compared to a control or reference level. For example, the quantity of a particular biomarker may be present at an elevated amount or at a decreased amount in samples of patients with a neoplastic disease compared to a reference level. In one embodiment, a “difference of a level” may be a difference between the quantity of a particular biomarker present in a sample as compared to a control of at least about 1%, at least about 2%, at least about 3%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 75%, at least about 80% or more. In one embodiment, a “difference of a level” may be a statistically significant difference between the quantity of a biomarker present in a sample as compared to a control. For example, a difference may be statistically significant if the measured level of the biomarker falls outside of about 1.0 standard deviations, about 1.5 standard deviations, about 2.0 standard deviations, or about 2.5 stand deviations of the mean of any control or reference group.

By “isolated”, when referring to a nucleic acid (e.g., an oligonucleotide such as RNA, DNA, or a mixed polymer) is meant a nucleic acid that is apart from a substantial portion of the genome in which it naturally occurs and/or is substantially separated from other cellular components which naturally accompany such nucleic acid. For example, any nucleic acid that has been produced synthetically (e.g., by serial base condensation) is considered to be isolated. Likewise, nucleic acids that are recombinantly expressed, cloned, produced by a primer extension reaction (e.g., PCR), or otherwise excised from a genome are also considered to be isolated. In some embodiments, the nucleic acid is isolated from the skin sample before further processing, such as PCR.

The term “label” as used herein, refers to any physical molecule directly or indirectly associated with a specific binding agent or antigen which provides a means for detection for that antibody or antigen. A “detectable label” as used herein refers any moiety used to achieve signal to measure the amount of complex formation between a target and a binding agent. These labels are detectable by spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, such as fluorescence, chemifluoresence, or chemiluminescence, electrochemiluminescence or any other appropriate means. Suitable detectable labels include fluorescent dye molecules or fluorophores.

As used herein interchangeably, a “microRNA,” “miR,” or “miRNA” refers to the unprocessed or processed RNA transcript from a miRNA gene. MicroRNAs (miRNAs) are non-coding RNAs of 19-25 nucleotides in length that regulate gene expression by inducing translational inhibition or cleavage of their target mRNA through base pairing to partially or fully complementary sites. The unprocessed miRNA gene transcript is also called a “miRNA precursor,” and typically comprises an RNA transcript of about 70-100 nucleotides in length. The miRNA precursor can be processed by digestion with an RNAse (for example, Dicer, Argonaut, or RNAse III) into an active 19-25 nucleotide RNA molecule. This active 19-25 nucleotide RNA molecule is also called the “processed” miRNA gene transcript or “mature” miRNA.

As used herein, “nucleic acid” refers broadly to segments of a chromosome, segments or portions of DNA, cDNA, and/or RNA. Nucleic acid may be derived or obtained from an originally isolated nucleic acid sample from any source (e.g., isolated from, purified from, amplified from, cloned from, or reverse transcribed from sample DNA or RNA).

As used herein, the term “oligonucleotide” refers to a short polymer composed of deoxyribonucleotides, ribonucleotides or any combination thereof. Oligonucleotides are generally between about 10 and about 100 nucleotides in length. Oligonucleotides are typically 15 to 70 nucleotides long, with 20 to 26 nucleotides being the most common. An oligonucleotide may be used as a primer or as a probe. An oligonucleotide is “specific” for a nucleic acid if the oligonucleotide has at least 50% sequence identity with a portion of the nucleic acid when the oligonucleotide and the nucleic acid are aligned. An oligonucleotide that is specific for a nucleic acid is one that, under the appropriate hybridization or washing conditions, is capable of hybridizing to the target of interest and not substantially hybridizing to nucleic acids which are not of interest. Higher levels of sequence identity are preferred and include at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity.

As used herein, a “primer” for amplification is an oligonucleotide that specifically anneals to a target or marker nucleotide sequence. The 3′ nucleotide of the primer should be identical to the target or marker sequence at a corresponding nucleotide position for optimal primer extension by a polymerase. As used herein, a “forward primer” is a primer that anneals to the anti-sense strand of double stranded DNA (dsDNA). A “reverse primer” anneals to the sense-strand of dsDNA.

As used herein, the term “reference level” refers to a level of a substance which may be of interest for comparative purposes. In one embodiment, a reference level may be the miRNA levels expressed as an average of the level of miRNA from an area of normal skin or skin containing nevi and not melanoma. Nucleic acid samples may also be normalized relative to an internal control nucleic acid.

As used herein, the term “sample” refers to a skin biopsy from the subject, such as would typically be used for histopathological examination, or any section derived from such a sample. That is, a suspected melanoma may be entirely excised from the skin, but the biopsy is fixed and embedded in paraffin, and sectioned for further examination.

As used herein, the term “subject” refers to a mammal, such as a human, but can also be another animal such as a domestic animal (e.g., a dog, cat, or the like), a farm animal (e.g., a cow, a sheep, a pig, a horse, or the like) or a laboratory animal (e.g., a monkey, a rat, a mouse, a rabbit, a guinea pig, or the like). The term “patient” refers to a subject who is, or is suspected to be, afflicted with melanoma.

The phrase “substantially the same as” in reference to a comparison of one value to another value for the purposes of clinical management of a disease or disorder means that the values are statistically not different. Differences between the values can vary, for example, one value may be within 20%, within 10%, or within 5% of the other value.

As used herein, the term “diagnostic score” refers to a single number or score, based on a statistical analysis of the measured level of one or more biomarkers that reflects a relationship of a specific subject to any one particular group of individuals, such as normal individuals or individuals having a disease or any progressive state thereof. In some embodiments, the diagnostic score is derived from a quantitative multivariate analysis, which reflects the overall statistical assessment of an individual patient's clinical condition based upon an integrated statistical calculation of a plurality of qualitatively unique factors, e.g., levels of diagnostic miRNA, combined with clinical presentation, etc.

Melanoma and Nevi

Nevus (or naevus, plural nevi or naevi, from nævus, Latin for “birthmark”) is the medical term for sharply-circumscribed and chronic lesions of the skin. These lesions are commonly named birthmarks and moles. Nevi are benign by definition.

A melanocytic nevus (nevomelanocytic nevus, nevocellular nevus) is a benign proliferation of melanocytes, and are very common; almost all adults have at least one, usually more. A melanocytic nevus may be congenital or acquired.

A dysplastic nevus usually an acquired melanocytic nevus with abnormal features making it difficult to distinguish from a melanoma. It can be a marker for an individual at risk for developing melanomas.

Melanoma is a malignant tumor of melanocytes. Melanocytes predominantly occur in skin, between the outer layer of the skin (the epidermis) and the next layer (the dermis), but are also found in other parts of the body, including the bowel and the eye (sec uveal melanoma). Melanoma can occur in any part of the body that contains melanocytes. Melanoma is less common than other skin cancers but is much more dangerous and causes the majority (75%) of deaths related to skin cancer.

Melanoma arises from DNA damage to melanocytcs. The early stage of the disease is called the radial growth phase, and the tumour is less than 1 mm thick. Next is the invasive radial growth phase, when individual cells start to acquire invasive potential. The Breslow's depth of the lesion is usually less than 1 mm (0.04 in), the Clark level is usually 2. The following step is invasive melanoma, “vertical growth phase” (VGP). The tumour attains invasive potential, growing into the surrounding tissue and can spread around the body through blood or lymph vessels to form metastases. The tumour thickness is usually more than 1 mm (0.04 in), and the tumour involves the deeper parts of the dermis.

An immunological reaction against the tumour during the VGP may be judged by the presence and activity of the tumour infiltrating lymphocytes (TILs). These cells sometimes completely destroy the primary tumour, this is called regression, which is the latest stage of the melanoma development. In certain cases, the primary tumour is completely destroyed and only the metastatic tumour is discovered.

Melanoma may also have a genetic predisposition. Mutations in CDKN2A, CDK4, MC1R, MDM2 SNP309 and those associated with xeroderma pigmentosum (XP) predispose one to melanoma. Familial melanoma is genetically heterogeneous,[10] and loci for familial melanoma have been identified on the chromosome arms 1p, 9p and 12q. Multiple genetic events have been related to the pathogenesis (disease development) of melanoma.

Clinical and Pathological Diagnosis

Melanoma is usually first detected by visual examination of the skin, notably (A) asymmetry, (B) a border that is uneven, ragged, or notched, (C) coloring of different shades of brown, black, or tan and (D) diameter that had changed in size. Normal moles are symmetrical, have an even border, even color, and no change in diameter. The main concern is distinguishing between a benign nevus, a dysplastic nevus, and a melanoma. Moles that are irregular in color or shape are often treated as candidates of melanoma. Following a visual examination and a dermatoseopic exam, or in vivo diagnostic tools such as a confocal microscope, a sample (biopsy) of the suspicious mole may be obtained.

Sample Preparation

When an atypical mole has been identified, a skin biopsy takes place in order to best diagnose it. Local anesthetic is used to numb the area, then the mole is biopsied. The biopsy material is then sent to a laboratory to be evaluated by a pathologist. A skin biopsy can be a punch, shave, or complete excision. The complete excision is the preferred method, but a punch biopsy can suffice if the patient has cosmetic concerns (i.e. the patient does not want a scar) and the lesion is small. A scoop or deep shave biopsy is often advocated, but should be avoided due to risk of a recurrent nevus, which can complicate future diagnosis of a melanoma, and the possibility that resulting scar tissue can obscure tumor depth if a melanoma is found to be present and re-excised.

Most dermatologists and dermatopathologists use a system devised by the NIH for classifying melanocytic lesions. In this classification, a nevus can be defined as benign, having atypia, or being a melanoma. A benign nevus is read as (or understood as) having no cytologic or architectural atypia. An atypical mole is read as having architectural atypia, and having (mild, moderate, or severe) cytologic (melanocytic) atypia. Usually, cytologic atypia is of more important clinical concern than architectural atypia. Usually, moderate to severe cytologic atypia will require further excision to make sure that the surgical margin is completely clear of the lesion.

The most important aspect of the biopsy report is that the pathologist indicates if the margin is clear (negative or free of melanocytic nevus), or if further tissue (a second surgery) is required. If this is not mentioned, usually a dermatologist or clinician will require further surgery if moderate to severe cytologic atypia is present—and if residual nevus is present at the surgical margin.

miRNA Markers to Distinguish Nevi from Melanoma

Distinguishing nevi from melanoma requires a high degree of skill. Misdiagnosis of a melanoma as a nevus can result in delay in treatment, which can be lethal because melanoma is an aggressive cancer that requires prompt intervention. Conversely, incorrectly identifying a nevus as a melanoma may subject a patient to aggressive treatment that is unnecessary and harmful. The present inventors have established that melanoma may be distinguished from nevi by monitoring the levels of select miRNA.

The methods described herein can distinguish melanoma, normal skin, nevi and malignant melanoma. Most importantly, the method is suitable for differentiating nevi from melanoma, and therefore fill a need for diagnosis that it not fully met by histology. As such, the methods of the invention can replace, supplement, or confirm histology. A particular advantage of the methods of the invention is that they provide independent objective evidence.

An additional advantage of the invention is that they can be performed on formalin fixed paraffin embedded (FFPE) tissue, and therefore can be used on the same samples that are processed for standard histopathological examination, and thus do not require a separate sample, or special handling. Another advantage is that the inventors have found that the miRNAs are stable in FFPE tissue and can be detected some time after fixing and embedding.

Another advantage of the invention is that the mRNA's chosen do not require a relatively pure sample of melanoma cells, and can detect melanoma in a sample that also contains normal skin, nevi and other skin cells. Thus, the miRNA assay is not overly sensitive to contamination nor require special handling beyond that which is normally used for preparation of FFPE tissue for regular histology.

The inventors have identified 50 miRNAs that can readily distinguish melanoma from nevi. Through statistical modeling and analysis, groups of the best 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14 markers have been identified. An increase in the number of markers used may improve sensitivity and accuracy, but comes with increased cost and complexity.

Additional diagnostic markers may be combined with the miRNA measurements to further aid diagnosis. For example, the clinical and/or histopathological results can be converted into a score, which is then combined with a score derived from the miRNA data. The combination of scores can be used to obtain a single “diagnostic score” that reflects the likelihood of melanoma.

Nucleic Acid Extraction and Detection

The level of a miRNA gene product in a sample can be measured using any technique that is suitable for detecting RNA expression levels in a biological sample. Suitable techniques for determining RNA expression levels in a biological sample are well known to those of skill in the art. These include, for example, Northern blot analysis, RT-PCR, and in situ hybridization.

The nucleic acid to be detected may be from a biological sample such as a tissue sample and the like. Various methods of extraction are suitable for isolating the DNA or RNA. Suitable methods include phenol and chloroform extraction. See Maniatis et al., Molecular Cloning, A Laboratory Manual, 2d, Cold Spring Harbor Laboratory Press, pp. 16-54 (1989). Numerous commercial kits also yield suitable DNA and RNA including, but not limited to, QIAamp™ mini blood kit, Agencourt Genfind™, Roche Cobas® Roche MagNA Pure® or phenol:chloroform extraction using Eppendorf Phase Lock Gels®, and the NucliSens extraction kit (Biomerieux, Marcy l'Etoile, France). In an illustrative embodiment, RNA is isolated from patient serum on the NucliSens easyMAG system (Biomeriux SA, France) according to the manufacturer's protocol.

In one embodiment, the level of at least one miRNA gene product is detected using Northern blot analysis. For example, total RNA can be purified from a sample in the presence of nucleic acid extraction buffer, followed by centrifugation. Nucleic acids are precipitated, and DNA is removed by treatment with DNase and precipitation. The RNA molecules are then separated by gel electrophoresis on agarose gels according to standard techniques, and transferred to nitrocellulose filters. The RNA is then immobilized on the filters by heating. Detection and quantification of specific RNA is accomplished using appropriately labeled DNA or RNA probes complementary to the RNA in question. See, for example, Molecular Cloning: A Laboratory Manual, J. Sambrook et al., eds., 2nd edition, Cold Spring Harbor Laboratory Press, 1989, Chapter 7.

Suitable probes (e.g., DNA probes or RNA probes) for Northern blot hybridization of a given miRNA gene product can be produced from the known nucleic acid sequences and include, but are not limited to, probes having at least about 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% complementarity to a miRNA gene product of interest, as well as probes that have complete complementarity to a miRNA gene product of interest. Methods for preparation of labeled DNA and RNA probes, and the conditions for hybridization thereof to target nucleotide sequences, are described in Molecular Cloning: A Laboratory Manual, J. Sambrook et al., eds., 2nd edition. Cold Spring Harbor Laboratory Press, 1989, Chapters 10 and 11.

For example, the nucleic acid probe can be labeled with, e.g., a radionuclide, such as ³H, ³²P, ³³P ¹⁴C, or ³⁵S; a heavy metal; a ligand capable of functioning as a specific binding pair member for a labeled ligand (e.g., biotin, avidin or an antibody); a fluorescent molecule; a chemiluminescent molecule; an enzyme or the like. Probes can be labeled to high specific activity by either the nick translation method or by the random priming method. Autoradiographic detection of hybridization can then be performed by exposing hybridized filters to photographic film. Densitometric scanning of the photographic films exposed by the hybridized filters provides an accurate measurement of miRNA levels. Using another approach, miRNA gene transcript levels can be quantified by computerized imaging systems.

In one embodiment, the miRNA is detected using a nucleic acid amplification process. Nucleic acid extracted from a sample can be amplified using nucleic acid amplification techniques well known in the art. By way of example, but not by way of limitation, these techniques can include the polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), nested PCR, ligase chain reaction. See Abravaya, K., et al., Nucleic Acids Research, 23:675-682, (1995), branched DNA signal amplification, Urdea, M. S., et al., AIDS, 7 (suppl 2):S11-S14, (1993), amplifiable RNA reporters, Q-beta replication, transcription-based amplification, boomerang DNA amplification, strand displacement activation, cycling probe technology, isothermal nucleic acid sequence based amplification (NASBA). See Kievits, T. et al., J Virological Methods, 35:273-286, (1991), Invader Technology, or other sequence replication assays or signal amplification assays may also be used.

Some methods employ reverse transcription of RNA to cDNA. The method of reverse transcription and amplification may be performed by previously published or recommended procedures. Various reverse transcriptases may be used, including, but not limited to, MMLV RT, RNase H mutants of MMLV RT such as Superscript and Superscript II (Life Technologies, GIBCO BRL, Gaithersburg, Md.), AMV RT, and thermostable reverse transcriptase from Thermus thermophilus. For example, one method which may be used to convert RNA to cDNA is the protocol adapted from the Superscript II Preamplification system (Life Technologies, GIBCO BRL, Gaithersburg, Md.; catalog no. 18089-011), as described by Rashtchian, A., PCR Methods Applic., 4:S83-S91, (1994).

In a suitable embodiment, PCR is used to amplify a target sequence of interest, PCR is a technique for making many copies of a specific template DNA sequence. The reaction consists of multiple amplification cycles and is initiated using a pair of primer sequences that hybridize to the 5′ and 3′ ends of the sequence to be copied. The amplification cycle includes an initial denaturation, and typically up to 50 cycles of annealing, strand elongation and strand separation (denaturation). In each cycle of the reaction, the DNA sequence between the primers is copied. Printers can bind to the copied DNA as well as the original template sequence, so the total number of copies increases exponentially with time. PCR can be performed as according to Whelan et al., J of Clin Micro, 33(3):556-561 (1995). Briefly, a PCR reaction mixture includes two specific primers, dNTPs, approximately 0.25 U of Taq polymerase, and 1×PCR Buffer.

The skilled artisan is capable of designing and preparing primers that are appropriate for amplifying a target or marker sequence. The length of the amplification primers depends on several factors including the nucleotide sequence identity and the temperature at which these nucleic acids are hybridized or used during in vitro nucleic acid amplification. The considerations necessary to determine a preferred length for an amplification primer of a particular sequence identity are well-known to a person of ordinary skill. For example, the length of a short nucleic acid or oligonucleotide can relate to its hybridization specificity or selectivity.

In some embodiments, the amplification may include a labeled primer or probe, thereby allowing detection of the amplification products corresponding to that primer or probe. In particular embodiments, the amplification may include a multiplicity of labeled primers or probes; such primers may be distinguishably labeled, allowing the simultaneous detection of multiple amplification products. Oligonucleotide probes can be designed which are between about 10 and about 100 nucleotides in length and hybridize to the amplified region. Oligonucleotides probes are preferably 12 to 70 nucleotides; more preferably 15-60 nucleotides in length; and most preferably 15-25 nucleotides in length. The probe may be labeled.

In one embodiment, a primer or probe is labeled with a fluorogenic reporter dye that emits a detectable signal. While a suitable reporter dye is a fluorescent dye, any reporter dye that can be attached to a detection reagent such as an oligonucleotide probe or primer is suitable for use in the invention. Such dyes include, but are not limited to, Acridine, AMCA, BODIPY, Cascade Blue, Cy2, Cy3, Cy5, Cy7, Edans, Eosin, Erythrosin, Fluorescein, 6-Fam, Tet, Joe, Hex, Oregon Green, Rhodamine, Rhodol Green, Tamra, Roz, and Texas Red.

In yet another embodiment, the detection reagent may be further labeled with a quencher dye such as Tamra, Dabcyl, or Black Hole Quencher® (BHQ), especially when the reagent is used as a self-quenching probe such as a TaqMan® (U.S. Pat. Nos. 5,210,015 and 5,538,848) or Molecular Beacon probe (U.S. Pat. Nos. 5,118,801 and 5,312,728), or other stemless or linear beacon probe (Livak et al., 1995, PCR Method Appl., 4:357-362; Tyagi et al, 1996, Nature Biotechnology, 14:303-308; Nazarenko et al., 1997, Nucl. Acids Res., 25:2516-2521; U.S. Pat. Nos. 5,866,336 and 6,117,635).

Nucleic acids may be amplified prior to detection or may be detected directly during an amplification step (i.e., “real-time” methods). For example, amplified fragments may be detected using standard gel electrophoresis methods. In some embodiments, amplified fractions are separated on an agarose gel and stained with ethidium bromide by methods known in the art to detect amplified fragments. In some embodiments, the target sequence is amplified using a labeled primer such that the resulting amplicon is detectably labeled. In some embodiments, the primer is fluorescently labeled.

In one embodiment, detection of a miRNA, such as a nucleic acid from an a miR-16 or miR-199a, is performed using the TaqMan® assay, which is also known as the 5′ nuclease assay (U.S. Pat. Nos. 5,210,015 and 5,538,848). The TaqMan® assay detects the accumulation of a specific amplified product during PCR. The TaqMan® assay utilizes an oligonucleotide probe labeled with a fluorescent reporter dye and a quencher dye. The reporter dye is excited by irradiation at an appropriate wavelength, it transfers energy to the quencher dye in the same probe via a process called fluorescence resonance energy transfer (FRET). When attached to the probe, the excited reporter dye does not emit a signal. The proximity of the quencher dye to the reporter dye in the intact probe maintains a reduced fluorescence for the reporter. The reporter dye and quencher dye may be at the 5′ most and the 3′ most ends, respectively or vice versa. Alternatively, the reporter dye may be at the 5′ or 3′ most end while the quencher dye is attached to an internal nucleotide, or vice versa. In yet another embodiment, both the reporter and the quencher may be attached to internal nucleotides at a distance from each other such that fluorescence of the reporter is reduced.

During PCR, the 5′ nuclease activity of DNA polymerase cleaves the probe, thereby separating the reporter dye and the quencher dye and resulting in increased fluorescence of the reporter. Accumulation of PCR product is detected directly by monitoring the increase in fluorescence of the reporter dye. The DNA polymerase cleaves the probe between the reporter dye and the quencher dye only if the probe hybridizes to the target-containing template which is amplified during PCR.

TaqMan® primer and probe sequences can readily be determined using the nucleic acid sequence information of the miRNA of interest. A number of computer programs, such as Primer Express (Applied Biosystems, Foster City, Calif.), can be used to rapidly obtain optimal primer/probe sets. It will be apparent to one of skill in the art that such primers and probes for detecting the target nucleic acids are useful in diagnostic assays for neoplastic disorders, such as HCC, and can be readily incorporated into a kit format. The present invention also includes modifications of the TaqMan® assay well known in the art such as the use of Molecular Beacon probes (U.S. Pat. Nos. 5,118,801 and 5,312,728) and other variant formats (U.S. Pat. Nos. 5,866,336 and 6,117,635).

In an illustrative embodiment, real time PCR is performed using TaqMan® Assays in combination with a suitable amplification/analyzer such as the ABI Prism® 7900HT Sequence Detection System. The ABI PRISM® 7900HT Sequence Detection System is a high-throughput real-time PCR system that detects and quantitates nucleic acid sequences. Real-time detection on the ABI Prism 7900HT or 7900HT Sequence Detector monitors fluorescence and calculates Rn during each PCR cycle. The threshold cycle, or Ct value, is the cycle at which fluorescence intersects the threshold value. The threshold value is determined by the sequence detection system software or manually. The Ct can be correlated to the initial amount of nucleic acids or number of starting cells using a standard curve.

In one embodiment, TaqMan® MicroRNA Assays are used to detect the miRNA. TaqMan® MicroRNA Assays are predesigned assays that are available for the majority of content found on the miRBase miRNA sequence repository. In another embodiment, the mirVana™ qRT-PCR miRNA Detection Kit (Ambion) is a used to detect and quantify the miRNA. This is a quantitative reverse transcription-PCR (qRT-PCR) kit enabling sensitive, rapid quantification of miRNA (miRNA) expression from total RNA samples.

As a quality control measure, an internal amplification control may be included in one or more samples to be extracted and amplified. The skilled artisan will understand that any detectable sequence that is not typically present in the sample can be used as the control sequence. A control sequence can be produced synthetically. If PCR amplification is successful, the internal amplification control amplicons can then be detected. Additionally, if included in the sample prior to purification of nucleic acids, the control sequences can also act as a positive purification control.

Statistical Methods

Statistical methods can be used to set thresholds for determining when the level in a subject can be considered to be different than or similar to a reference level. In addition, statistics can be used to determine the validity of the difference or similarity observed between a patient's circulating miRNA level and the reference level. Useful statistical analysis methods are described in L. D. Fisher & G. vanBelle, Biostatistics: A Methodology for the Health Sciences (Wiley-Interscience, NY, 1993). For instance, confidence (“p”) values can be calculated using an unpaired 2-tailed t test, with a difference between groups deemed significant if the p value is less than or equal to 0.05. As used herein a “confidence interval” or “CI” refers to a measure of the precision of an estimated or calculated value. The interval represents the range of values, consistent with the data that is believed to encompass the “true” value with high probability (usually 95%). The confidence interval is expressed in the same units as the estimate or calculated value. Wider intervals indicate lower precision; narrow intervals indicate greater precision. Preferred confidence intervals of the invention are 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% and 99.99%. A “p-value” as used herein refers to a measure of probability that a difference between groups happened by chance. For example, a difference between two groups having a p-value of 0.01 (or p=0.01) means that there is a 1 in 100 chance the result occurred by chance. Preferred p values are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, and 0.0001. Confidence intervals and p-values can be determined by methods well-known in the art. See, e.g., Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York, 1983.

On linear model for assessing differential expression in microarray experiments: Smith G K (2004) “Linear models and empirical bayes method for assessing differential expression in microarray experiments” Statistical Applications in Genetics and Molecular Biology. For AUC calculation: Mason S J and Graham N E (1982) “Areas beneath the relative operating characteristics (ROC) and relative operating levels (ROL) curves: Statistical significance and interpretation,” Q. J. R. Meteorol. Soc. textbf30 291-303. Multiple algorithms program for marker combination selection: An R based program with nine algorithms including random forest, ada boosting, svm, bagging, logistic regression, lasso, matt, cart, ctree is available, for example, as open-source software from the R Foundation. Random forests were also conducted according to Breiman, L. (2001), Random Forests, Machine Learning 45(1), 5-32. Sec also Breiman, L (2002), “Manual On Setting Up, Using, And Understanding Random Forests V3.1.

In connection with miRNA used to diagnose melanoma, one may seek levels that are lower or higher than a control. The term “elevated levels” or “higher levels” as used herein refers to levels of an miRNA that are higher than what would normally be observed in a comparable sample from control or normal subjects or normal tissue from the patient (i.e., a reference value). Similarly, “reduced levels” or “lower levels” as used herein refer to levels of that are lower than what would normally be observed in a comparable sample from control or normal subjects, or normal tissue from the patient (i.e., a reference value). In some embodiments, “control levels” (i.e., normal levels) refer to a range of miRNA levels that would be normally be expected to be observed in nevi, or normal skin. A control level may be used as a reference level for comparative purposes. The ranges accepted as outside “control levels” are dependent on a number of factors. For example, one laboratory may routinely determine the level of circulating miRNA in a sample that is different than the miRNA obtained for the same sample by another laboratory. Also, different assay methods may achieve different value ranges. Value ranges may also differ in various sample types, for example, different body fluids or by different treatments of the sample. One of ordinary skill in the art is capable of considering the relevant factors and establishing appropriate reference ranges for “control values” and “elevated/reduced values” of the present invention. For example, a series of samples from control subjects and subjects diagnosed with melanoma can be used to establish ranges that are “normal” or “control” levels and ranges that are “elevated” or “reduced” than the control range.

The level of one or more miRNAs measured in the test sample is normalized, such as by comparison to an internal reference nucleic acid, e.g., U44 or small RNA U6. The levels of the one or more miRNAs may then be compared to a reference value to determine if the levels of the one or more miRNAs are elevated or reduced relative to the reference value. Typically, the reference value is the level measured in a comparable sample from one or more healthy individuals. An increase or decrease in the level of the one or more miRNAs may be used in conjunction with clinical factors to diagnose melanoma.

In some embodiments, the level of one or more miRNAs is combined with one or more additional markers to improve diagnostic sensitivity and specificity. Exemplary markers include, but are not limited to, any useful diagnostic marker associated with melanoma including those which may be assessed by fluorescence in-situ hybridization (FISH) and/or comparative genomic hybridization (CGH).

Kits

A kit may be used for conducting the diagnostic and prognostic methods described herein. Typically, the kit should contain, in a carrier or compartmentalized container, reagents useful in any of the above-described embodiments of the diagnostic method. The carrier can be a container or support, in the form of e.g., bag, box, tube, rack, and is optionally compartmentalized. The carrier may define an enclosed confinement for safety purposes during shipment and storage. In one embodiment, the kit includes one or more PCR primers capable of amplifying miRNA selected from miR-132, miR-150, miR-339-5p, miR-15b, miR-342-3p, miR-572, miR-155, miR-425, miR-1202, miR-1268, HBII-382_s, miR-1225-5p, miR-30c, miR-106b-star, miR-125a-5p, mgU6-53B, miR-25, miR-149-star, miR-939, miR-92b-star, miR-500-star, miR-22, HBII-142_x, miR-181b, HBII-142, U38B, miR-663, miR-1224-5p, miR-23a, HBII-85-6_x, miR-1207-5p, miR-1301, miR-1228-star, miR-345, miR-30a-star, ENSG00000199411_s, ENSG00000202327, miR-92a, miR-127-3p, HBII-85-26, miR-1308, miR-31, miR-921, miR-146b-5p, miR-768-3p, miR-708, miR-139-5p, ACA24_x, miR-501-3p, miR-502-3p, miR-923, and miR-191.

In further embodiments, the invention comprise a kit. In one embodiment, a kit for differentially diagnosing between melanoma and nevus in a skin sample comprises primers for the amplification of at least two miRNA selected from the group consisting of miR-150, miR-149-star, miR-1308; miR-191; miR-1228-star; ENSG00000199411_s; miR-1268; miR-923; miR-23a: miR-132; miR-1207.5p; miR-342.3p; U38B; and miR-155.

In another embodiment, the kit comprises primers for the amplification of at least two miRNAs selected from the group consisting of: miR-1268, miR-1228-star, miR-92b-star, miR-155, miR-345, miR-425, miR-132, miR-1207-5p, miR-1301, miR-663, miR-339-5p, miR-149-star, miR-150, miR-18a, miR-103, miR-191, miR-296-3p, miR-31, miR-107*, miR-93*, miR-1275*, miR-181B*, miR-921*, miR-1225-5p, miR-1202, and miR-342-3p.

The kit may also primers for amplification of controls, such as miR-27b, miR-195, miR-199b-3p, and miR-199a-3p.

The kit may also include suitable buffers, reagents for isolating nucleic acid, and instructions for use. Kits may also include a microarray for measuring miRNA level.

The primers may be labeled with a detectable marker such as radioactive isotopes, or fluorescence markers. Instructions for using the kit or reagents contained therein are also included in the kit.

EXAMPLES

The present methods and kits, thus generally described, will be understood more readily by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present methods and kits. The following is a description of the materials and experimental procedures used in the example.

Example 1

137 samples were examined: (1) 19 normal skin; (2) 38 nevi, of which 20 were intradermal, where the nevus cells are located in the dermis only, and 18 were compound nevus, which is a mixture of junctional and intradermal proliferation and are slightly raised and brown to black; (3) 58 primary melanoma; (4) 22 metastatic melanoma.

Total RNA, including low molecular weight RNA, was isolated from ten 10 μm-FFPE sections with RecoverAll RNA extraction kit (Applied Biosystems), according to the manufacturer's protocol.

The Affymetrix GeneChip® miRNA array was used, according to the manufacturer's protocol, to evaluate miRNA expression in FFPE samples. The miRNA array covers 71 organisms, including human, mouse, rat, and monkey, and contains 1801 sets of human miRNA, snoRNAs and scaRNAs. Samples were labeled using the Genisphere FlashTag™ Biotin Labeling Assay, which utilizes the 3DNA™ technology. The 3DNA™ dendrimer was ligated to samples to allow multiple biotin molecules (˜15) to bind to each poly-A tailed RNA molecule. Following FlashTag™ ligation, samples were hybridized on the Affymetrix GeneChip® miRNA array overnight. The hybridized chips were washed and processed to scan in an Affimatrix GeneChip Scanner 3000 7G.

Statistical Analyses

The raw microarray data was analyzed using the miRNA QC tool which performed an RNA normalization and extracted signals for data analysis. Log 2 values were used for miRNA expression in each group. The log 2 fold change (log 2 FC) was calculated by subtracting the mean of log 2 of group 1 from the mean log 2 of group 2. Student's t test was used to compare the miRNA expression level of each miRNA between normal skin, nevi, melanoma, and metastatic melanoma. P<0.05 was used as statistical significance. The area under ROC (AUC) was calculated to reflect the separation between each group. The default AUC value is 0.5 meaning no separation, and the maximum possible value is 1.0, meaning complete separation between each group.

A screen on probe signal detection in samples was performed having of 137 samples at least >30 samples with signal detectable. There are 729 of 1801 human probes satisfying such condition. These 729 candidate markers were analyzed using multiple algorithm programs.

Two or three markers completely separate these melanoma from nevi, as confirmed by multiple algorithms. The best 15 miRNA markers from randomForest share 14 markers overlap with the best 15 markers from boosting. In addition, these 14 best overlap markers include 6 of 7 best markers from previous analyses of 40 melanoma vs 20 nevi. These results therefore show consistency across different assays at different times.

Example 2

Additional experiments were performed to identify miRNA that can distinguish between different skin conditions.

For melanoma vs nevus, specimens (n=380) included a training set of 20 paraffin-embedded blocks of normal skin, 60 paraffin-embedded blocks of skin biopsies with benign nevus, 60 paraffin-embedded blocks of skin biopsies with malignant melanoma. Next, a validation set of 100 paraffin-embedded blocks of skin biopsies with benign nevus, 100 paraffin-embedded blocks of skin biopsies with malignant melanoma and 50 paraffin-embedded blocks of dysplastic nevus.

To distinguish between primary melanoma and metastatic melanoma, 180 study specimens included a training set of 60 paraffin-embedded blocks of skin biopsies with malignant melanoma and 30 paraffin-embedded blocks of metastatic melanoma. The validation set comprised 60 paraffin-embedded blocks of skin biopsies with malignant melanoma and 30 paraffin-embedded blocks of metastatic melanoma.

The subject population targeted all ethnicities and was approximately 50% male and 50% female. Specimens of adults 18 year and younger, and all subjects 89 years or older (“>90”) were discarded.

Tissues (normal benign, nevi and indeterminate nevus, malignant melanoma and metastatic melanoma) were collected at DermaPath. 240 nevi and 40 melanoma FFPEs were purchased from BioTheme.

Total RNA, including low molecular weight RNA, was isolated from ten 10 μm-FFPE sections with RecoverAll RNA extraction kit from Applied Biosystems according to the manufacturer's protocol.

The Affymetrix GeneChip® miRNA array was used to evaluate miRNA expression in FFPE samples. The miRNA array covers 71 organisms, including human, mouse, rat, and monkey, and contains 1801 sets of human miRNA, snoRNAs and scaRNAs.

The experiment procedure was according to the Affymetrix miRNA expression analysis manual. Samples were labeled with the Genisphere FlashTag™ Biotin Labeling Assay, which utilizes the 3DNA™ technology. The 3DNA™ dendrimer was ligated to samples to allow multiple biotin molecules (˜15) to bind to each poly-A tailed RNA molecule. Following FlashTag™ ligation, samples were hybridized on the Affymetrix GeneChip® miRNA array overnight. The hybridized chips were washed and processed to scan in an Affimatrix GeneChip Scanner 3000 7G.

Statistical Analyses

Log 2 values were used for miRNA expression in each group. The log 2 fold change (log 2 FC) was calculated by subtracting the mean of log 2 of group 1 from the mean log 2 of group 2. Student's t test was used to compare the miRNA expression level of each miRNA between normal skin, nevi, melanoma, and metastatic melanoma. P<0.05 was used as statistical significance. The area under ROC (AUC) was calculated to reflect the separation between each group. The default AUC value is 0.5 meaning no separation, and the maximum possible value is 1.0, meaning complete separation between each group.

Semi-Quantitative Reverse-Transcriptase PCR Analysis of miRNA

Two-step TaqMan reverse-transcriptase PCR analysis was performed for analysis of miRNAs. Reverse transcription was performed in a 15-μl reaction volume using specific primers for each miRNA contained in the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Foster City, Calif.) by sequentially incubating at 16° C. for 30 min, 42° C. for 30 min, and 85° C. for 5 min. Real-time PCR was done using the standard TaqMan MicroRNA assay protocol on an Applied Biosysytems 7900 system (Applied Biosystems). Each PCR mixture (20 μl) included the reverse transcription products, TaqMan 2× Universal PCR Master Mix without UNG Amperase, miRNA-specific TaqMan probes, and primers supplied by Applied Biosystems. The reactions were incubated in a 96-well plate with an initial denaturation at 95° C. for 10 min, followed by 40 cycles of 95° C. for 15 s and 60° C. for 1 min. The level of miRNA expression was measured using the threshold cycle (Ct), the fractional cycle number at which the fluorescence of each sample passes a fixed threshold. miRNA expression levels were normalized using an endogenous small RNA control U44 (Applied Biosystems). The expression of miRNA relative to small RNA U44 is reported as ΔCt, which was calculated by subtracting the Ct of U44 RNA from the Ct of target miRNA.

4. Results

MiRNA expression was analyzed between each group, Table 1 shows 523 miRNAs which have significant expression level, either over or less expression, between melanoma and nevi groups (p<0.05). Table 2 showed 50 miRNAs with the most significant expression between the two groups. Table 3 showed 378 miRNAs which have significant expression differences between melanoma and normal skin groups (p<0.05). Table 4 showed 50 miRNAs with the most significant expression differences between the two groups. Table 5 showed 174 miRNAs which have significant expression differences between melanoma and metastatic melanoma groups (p<0.05). Table 6 showed 50 miRNAs with the most significant expression differences between the two groups. Table 7 showed 442 miRNAs which have significant expression differences between nevi and normal skin groups (p<0.05). Table 8 showed 50 miRNAs with the most significant expression differences between the two groups.

TABLE 1 miRNAs significantly expressed between melanoma and nevi Log2- #Sample Probe Name FC P value AUC ProbeType Detected miR-132 2.98  1.2728E−32 0.994 miRNA 96 miR-150 3.27 5.83192E−30 1.000 miRNA 117 miR-339-5p 2.73 1.69388E−27 0.988 miRNA 75 miR-15b 3.02 6.41784E−27 0.978 miRNA 125 miR-342-3p 2.23 7.03798E−26 0.993 miRNA 136 miR-572 −2.78 1.00008E−25 0.970 miRNA 116 miR-155 4.14 1.28454E−25 0.981 miRNA 123 miR-425 2.74 8.13135E−25 0.975 miRNA 114 miR-1202 −2.63 2.97795E−23 0.986 miRNA 57 miR-1268 −2.68 6.05905E−23 0.997 miRNA 133 HBII-382_s −1.71 6.58931E−22 0.971 scaRna 126 miR-1225-5p −2.36 1.05361E−21 0.953 miRNA 90 miR-30c 2.39 2.58594E−21 0.980 miRNA 125 miR-106b-star 2.24 3.48507E−21 0.961 miRNA 72 miR-125a-5p 2.39 6.90609E−21 0.967 miRNA 128 mgU6-53B −1.51 1.74782E−20 0.973 CDBox 99 miR-25 2.57 1.52769E−19 0.956 miRNA 118 miR-149-star −2.03 1.64858E−19 1.000 miRNA 135 miR-939 −2.21  1.9129E−19 0.973 miRNA 57 miR-92b-star −2.30 2.05085E−19 0.969 miRNA 111 miR-500-star 2.38 3.11256E−19 0.956 miRNA 97 miR-22 2.69  3.8844E−19 0.965 miRNA 120 HBII-142_x −1.22 4.93318E−19 0.981 CDBox 135 miR-181b 2.41 1.00639E−18 0.960 miRNA 130 HBII-142 −1.33 1.18794E−18 0.988 CDBox 135 U38B −1.95 1.35964E−18 0.975 CDBox 134 miR-663 −2.10 2.04672E−18 0.984 miRNA 134 miR-1224-5p −2.54 2.91169E−18 0.946 miRNA 78 miR-23a 1.18 2.99523E−18 0.967 miRNA 137 HBII-85-6_x −1.73 4.78418E−18 0.939 CDBox 137 miR-1207-5p −2.14 4.98991E−18 0.995 miRNA 133 miR-1301 2.36 5.22389E−18 0.931 miRNA 54 miR-1228-star −2.41 5.51513E−18 0.997 miRNA 134 miR-345 2.45 6.43552E−18 0.942 miRNA 75 miR-30a-star 2.32 6.92165E−18 0.932 miRNA 64 ENSG00000199411_s −1.97 7.23325E−18 0.991 snoRNA 135 ENSG00000202327 −1.40 8.75839E−18 0.938 snoRNA 54 miR-92a 1.67 9.49427E−18 0.979 miRNA 136 miR-127-3p 2.43 9.84615E−18 0.936 miRNA 89 HBII-85-26 −2.07 1.50619E−17 0.951 CDBox 136 miR-1308 −2.14 1.99888E−17 0.999 miRNA 135 miR-31 3.29 2.46697E−17 0.913 miRNA 103 miR-921 −1.46 2.61089E−17 0.928 miRNA 49 miR-146b-5p 2.37 6.10422E−17 0.918 miRNA 83 miR-768-3p −1.05 6.66153E−17 0.949 miRNA 137 miR-708 2.29 6.86187E−17 0.929 miRNA 102 miR-139-5p 2.23 2.78962E−16 0.922 miRNA 80 ACA24_x 1.33 3.37973E−16 0.925 HAcaBox 98 miR-501-3p 1.83 4.76751E−16 0.923 miRNA 80 miR-502-3p 2.16 5.19509E−16 0.925 miRNA 110 miR-923 −1.86 5.55191E−16 0.996 miRNA 135 U94 −1.17 6.40118E−16 0.926 CDBox 109 miR-574-3p 2.32 8.68105E−16 0.946 miRNA 122 miR-135a-star −1.90 1.03062E−15 0.914 miRNA 44 ENSG00000207098_x −1.03 1.25346E−15 0.924 snoRNA 86 U38B_x −1.55 2.54255E−15 0.958 CDBox 133 miR-423-3p 1.83 2.74508E−15 0.932 miRNA 107 miR-198 −1.79 3.57486E−15 0.909 miRNA 31 ACA16 1.43 4.07871E−15 0.919 HAcaBox 47 ACA25 1.06 4.62028E−15 0.910 HAcaBox 73 miR-769-5p 1.63 4.79679E−15 0.920 miRNA 56 ENSG00000212523_x −1.38 6.22717E−15 0.927 snoRNA 135 mgU6-53B_x −0.99 6.32629E−15 0.919 CDBox 121 Z17B 0.97  1.1349E−14 0.903 CDBox 117 U81_x 1.22 1.25935E−14 0.913 CDBox 120 miR-532-5p 2.10 1.27686E−14 0.933 miRNA 113 ENSG00000200879 1.27 1.49096E−14 0.917 snoRNA 120 HBII-419 −1.07 2.02184E−14 0.938 CDBox 133 U58B_x 0.96 9.25535E−14 0.898 CDBox 132 ENSG00000201619 −1.66 1.14582E−13 0.911 snoRNA 62 miR-28-3p 2.15 1.17402E−13 0.900 miRNA 94 miR-1300 −1.86 1.21666E−13 0.892 miRNA 32 miR-191 1.18  1.2767E−13 0.983 miRNA 135 miR-181a-2-star 1.93 2.91245E−13 0.898 miRNA 99 U38A −1.52 3.04032E−13 0.971 CDBox 134 U59A −1.01 3.86379E−13 0.948 CDBox 135 ENSG00000212397 −1.21 4.58111E−13 0.902 snoRNA 134 HBII-85-26_x −1.13 4.73092E−13 0.897 CDBox 135 miR-638 −1.62 5.93273E−13 0.967 miRNA 135 miR-421 1.84 6.09941E−13 0.881 miRNA 68 miR-21 2.62 6.26336E−13 0.888 miRNA 79 miR-24-2-star 1.81 8.95923E−13 0.879 miRNA 63 U36C −0.92  1.1248E−12 0.931 CDBox 135 miR-92b 1.43 1.15239E−12 0.901 miRNA 91 miR-199a-5p 1.69 1.22362E−12 0.901 miRNA 129 ACA24_s 1.36 1.86137E−12 0.880 HAcaBox 128 ACA9 1.36 1.92813E−12 0.888 HAcaBox 82 ENSG00000199411_x −0.87 2.22903E−12 0.868 snoRNA 132 ENSG00000199435 −0.94 2.45067E−12 0.903 snoRNA 63 miR-182 1.80 3.73608E−12 0.905 miRNA 113 miR-1275 −1.56 3.97629E−12 0.888 miRNA 127 miR-150-star −1.70 4.59839E−12 0.897 miRNA 48 ACA48_x 1.08 5.05927E−12 0.881 HAcaBox 130 HBII-85-8_x −1.09 5.47576E−12 0.877 CDBox 135 miR-99b 1.33 5.48764E−12 0.907 miRNA 129 U74_x −1.00 6.23625E−12 0.941 CDBox 135 miR-1271 1.71  6.2815E−12 0.885 miRNA 70 ENSG00000206637_x −0.86 6.43179E−12 0.878 snoRNA 59 miR-20b 2.16 1.13294E−11 0.892 miRNA 99 ENSG00000200652 −0.93 1.18064E−11 0.876 snoRNA 40 U13 −0.94 1.19458E−11 0.899 CDBox 135 ENSG00000201660 −1.13 1.27245E−11 0.903 snoRNA 132 HBII-85-23_x 1.18 1.36679E−11 0.883 CDBox 58 miR-149 1.85 1.38908E−11 0.896 miRNA 100 ACA9_x 1.14 2.05352E−11 0.877 HAcaBox 77 miR-1234 −1.10 2.16855E−11 0.856 miRNA 68 miR-1180 1.50 2.61371E−11 0.878 miRNA 37 miR-30a 1.80 3.39494E−11 0.861 miRNA 113 U44_x 1.13 3.71768E−11 0.953 CDBox 135 miR-181c 1.54 4.41605E−11 0.861 miRNA 59 ENSG00000202498_x −1.07 4.69703E−11 0.855 snoRNA 137 miR-940 −1.22 5.55177E−11 0.861 miRNA 53 miR-500 1.76 6.23662E−11 0.854 miRNA 86 ENSG00000212627 −0.78 6.92771E−11 0.871 snoRNA 69 ENSG00000207027 −0.84 8.23933E−11 0.879 snoRNA 36 miR-27a-star 1.63 8.66657E−11 0.856 miRNA 40 miR-128 1.66 9.46068E−11 0.858 miRNA 44 snR38C −1.01 9.50785E−11 0.926 CDBox 134 ENSG00000212266 −0.93  9.6633E−11 0.848 snoRNA 93 miR-185 1.76 1.06661E−10 0.922 miRNA 132 ACA64 −0.97 1.08821E−10 0.858 HAcaBox 33 miR-151-3p 1.71 1.37187E−10 0.884 miRNA 117 miR-130b 2.12 1.55732E−10 0.870 miRNA 90 miR-27b-star 1.70 1.72122E−10 0.849 miRNA 51 miR-665 −1.43 1.78835E−10 0.853 miRNA 47 miR-18a 2.01 1.91068E−10 0.854 miRNA 77 ACA36_x −1.03  2.0756E−10 0.875 HAcaBox 88 ENS00000212432_s −0.94 2.08962E−10 0.882 snoRNA 75 ENSG00000202093_x 1.01 4.04338E−10 0.873 snoRNA 125 miR-487b 1.41 4.22069E−10 0.848 miRNA 58 U65 −0.98 4.31315E−10 0.847 HAcaBox 128 miR-532-3p 1.19 4.73885E−10 0.881 miRNA 95 U101 −0.79 5.89958E−10 0.845 CDBox 134 miR-138 2.09 6.68933E−10 0.839 miRNA 82 HBII-99 1.01 7.89663E−10 0.847 CDBox 122 miR-222 1.12 9.21171E−10 0.913 miRNA 135 miR-652 1.46 9.82734E−10 0.862 miRNA 119 miR-10a 1.82 9.97674E−10 0.843 miRNA 69 miR-625 1.46 1.06062E−09 0.833 miRNA 59 ENSG00000212182 −0.89 1.13997E−09 0.828 snoRNA 59 miR-331-3p 1.47 1.22124E−09 0.834 miRNA 64 miR-1281 −1.46 1.31027E−09 0.843 miRNA 137 miR-28-5p 1.53 1.35526E−09 0.863 miRNA 105 ACA23 0.76 2.45172E−09 0.837 HAcaBox 113 U27 1.04 2.55698E−09 0.874 CDBox 134 miR-339-3p 1.12  2.9234E−09 0.836 miRNA 60 U68_x 1.00 3.99716E−09 0.861 HAcaBox 133 miR-92a-2-star −1.06 4.67209E−09 0.830 miRNA 46 miR-107 1.08 5.05068E−09 0.946 miRNA 135 U104 −0.79 5.07142E−09 0.918 CDBox 135 miR-671-5p −1.16 6.05404E−09 0.877 miRNA 69 miR-193b 1.22 6.27171E−09 0.852 miRNA 130 miR-1272 −1.13 6.29028E−09 0.826 miRNA 67 miR-221 1.23  6.5716E−09 0.911 miRNA 135 14qll-14 1.27  6.8516E−09 0.828 CDBox 56 miR-1288 −0.92 6.95468E−09 0.804 miRNA 43 ENSG00000212458 −0.80 7.62772E−09 0.853 snoRNA 95 miR-584 1.61 7.63622E−09 0.815 miRNA 57 U106 0.64 8.00299E−09 0.821 CDBox 112 miR-152 1.58 8.26552E−09 0.885 miRNA 122 ENSG00000212551 −0.72 9.08023E−09 0.841 snoRNA 73 U96a_x −0.89 1.00143E−08 0.863 CDBox 134 ENSG00000212139_x −0.74 1.22887E−08 0.811 snoRNA 124 miR-409-3p 1.14 1.25923E−08 0.832 miRNA 92 miR-214-star 1.59 1.34912E−08 0.830 miRNA 47 ACA20 0.91 1.36416E−08 0.873 HAcaBox 131 HBII-82 −0.73 1.52532E−08 0.823 CDBox 90 U49A −0.86 1.55769E−08 0.856 CDBox 135 miR-362-5p 1.63 1.67441E−08 0.824 miRNA 94 ACA40_x 1.17 1.68127E−08 0.872 HAcaBox 134 U63 −0.98 1.83203E−08 0.916 CDBox 135 HBII-436 0.76 1.85498E−08 0.810 CDBox 102 miR-15a 1.74  1.8639E−08 0.826 miRNA 80 U103_s 0.83 2.02002E−08 0.809 CDBox 111 miR-1185 −0.96 2.35573E−08 0.822 miRNA 42 ACA15_s 0.74 2.84086E−08 0.812 HAcaBox 116 miR-30b 1.37 3.22874E−08 0.843 miRNA 128 spike_in-control-21 −0.70 3.25874E−08 0.832 Oligonucleotide 42 spike-in controls U15B 0.71 3.51612E−08 0.825 CDBox 129 miR-143 1.99 3.72999E−08 0.820 miRNA 126 U67_x 0.79 4.12783E−08 0.813 HAcaBox 50 miR-10b 1.41 4.29563E−08 0.821 miRNA 106 U99 −0.65 4.50576E−08 0.817 HAcaBox 137 miR-194 1.27 5.18787E−08 0.813 miRNA 54 miR-324-5p 1.14 5.23291E−08 0.804 miRNA 102 miR-125b-2-star 1.19 5.54514E−08 0.819 miRNA 58 HBII-52-37_x −0.76 8.93997E−08 0.805 CDBox 37 ACA48 0.72 9.13165E−08 0.825 HAcaBox 121 miR-103 0.97 9.25296E−08 0.931 miRNA 135 miR-342-5p 1.32 9.27998E−08 0.827 miRNA 98 miR-93 1.08 9.93604E−08 0.896 miRNA 134 miR-296-3p −1.00 1.10115E−07 0.818 miRNA 90 miR-200b-star 1.30 1.13507E−07 0.791 miRNA 74 U44 0.87 1.17791E−07 0.897 CDBox 134 ENSG00000207016_x −0.63 1.23122E−07 0.803 snoRNA 48 spike_in-control-31 −0.14 1.24357E−07 0.798 Oligonucleotide 137 spike-in controls U68 0.83 1.72086E−07 0.856 HAcaBox 130 U24 −0.55 2.39582E−07 0.822 CDBox 133 U83 0.62 2.42808E−07 0.846 CDBox 135 ENSG00000212273_x −0.80 2.45728E−07 0.811 snoRNA 126 14qll-14_x 1.03 2.59298E−07 0.797 CDBox 56 miR-660 1.26 2.65402E−07 0.788 miRNA 71 14qll-7 −0.70 2.89281E−07 0.762 CDBox 77 U78_s 1.01 3.07849E−07 0.836 CDBox 135 miR-744 −1.03 3.11141E−07 0.818 miRNA 133 U22 −0.54 3.14269E−07 0.793 CDBox 135 ACA11 0.70 3.50307E−07 0.793 HAcaBox 48 ENSG00000212553_x −0.69 3.51184E−07 0.794 snoRNA 60 U67 0.76 3.78503E−07 0.796 HAcaBox 44 miR-382 1.35 3.83783E−07 0.787 miRNA 47 miR-212 1.58 3.89653E−07 0.854 miRNA 62 miR-93-star 1.26 4.02443E−07 0.808 miRNA 48 U18C_x 0.74  4.1571E−07 0.794 CDBox 74 miR-1274a 1.22  4.1577E−07 0.788 miRNA 48 ACA25_x 0.77 4.42948E−07 0.799 HAcaBox 114 U15A −0.74 4.57833E−07 0.821 CDBox 128 14ql-1 −0.77 5.08646E−07 0.784 CDBox 70 ENSG00000201816 −0.60 5.51521E−07 0.789 snoRNA 49 miR-145 1.56 6.30644E−07 0.775 miRNA 135 HBII-420 −0.75 7.96309E−07 0.828 CDBox 131 ACA2b −0.66 8.56351E−07 0.787 HAcaBox 47 miR-1273 −0.94 1.01224E−06 0.768 miRNA 47 miR-641 −0.85  1.0165E−06 0.765 miRNA 39 ENSG00000202216 −0.60 1.02726E−06 0.778 snoRNA 53 U97 −0.72 1.06795E−06 0.794 CDBox 133 EN8G00000200706_x −0.66 1.06937E−06 0.778 snoRNA 43 miR-363 1.33 1.20135E−06 0.806 miRNA 44 ENSG00000201348 −0.48 1.23741E−06 0.779 snoRNA 94 U83A 0.75 1.32496E−06 0.818 CDBox 127 miR-197 1.27  1.3431E−06 0.807 miRNA 103 miR-106b 1.23 1.49024E−06 0.805 miRNA 127 HBII-180A_x 0.60 1.62491E−06 0.784 CDBox 123 snR38B −0.76 1.68966E−06 0.803 CDBox 126 snR38A −0.87 1.99208E−06 0.838 CDBox 130 miR-134 1.15 2.09717E−06 0.774 miRNA 61 miR-29b-1-star 1.41 2.20964E−06 0.764 miRNA 46 mgh18S-121 −0.66 2.37986E−06 0.833 CDBox 134 mgU6-77 0.59 2.38866E−06 0.742 CDBox 129 HBII-52-25_x −0.60 2.44738E−06 0.767 CDBox 57 U48 0.83 2.51688E−06 0.777 CDBox 129 14qll-19 −0.73 2.52816E−06 0.770 CDBox 43 miR-552 −0.66 2.70576E−06 0.760 miRNA 34 miR-424-star 1.31 2.79344E−06 0.762 miRNA 41 HBII-316 −0.60 3.06016E−06 0.795 CDBox 130 U35B −0.64 3.21695E−06 0.811 CDBox 128 miR-30d 1.02 3.25088E−06 0.817 miRNA 131 miR-1248 −0.72 3.27937E−06 0.746 miRNA 39 ENSG00000212579_x −0.65 3.55239E−06 0.758 snoRNA 100 hsa-let-7b 0.90 3.76667E−06 0.919 miRNA 136 miR-29b-2-star −0.78 3.86015E−06 0.760 miRNA 126 miR-148a 1.04 4.16979E−06 0.773 miRNA 41 miR-629 1.16 4.35623E−06 0.776 miRNA 64 HBII-295 −0.58 4.36224E−06 0.767 CDBox 128 miR-877 −1.00 4.43293E−06 0.794 miRNA 126 ACA62 −0.61 4.43469E−06 0.774 HAcaBox 88 ENSG00000207100_x −0.50 4.49659E−06 0.765 snoRNA 72 HBII-180C 0.62 4.78509E−06 0.780 CDBox 115 HBII-115 −0.57 5.03448E−06 0.768 CDBox 117 miR-192 1.14 5.73989E−06 0.763 miRNA 32 miR-505-star 1.23 5.74055E−06 0.762 miRNA 58 spike_in-control-7 −0.80 5.89851E−06 0.790 Oligonucleotide 54 spike-in controls miR-422a 1.17 5.96437E−06 0.782 miRNA 84 U83B −0.56 6.55847E−06 0.842 CDBox 135 ENSG00000212134_x −0.65 6.92676E−06 0.752 snoRNA 88 U50B_x −0.67 7.13703E−06 0.783 CDBox 135 miR-193a-3p 0.91 8.82408E−06 0.747 miRNA 42 ENSG00000201848 −0.62 9.39369E−06 0.759 snoRNA 34 miR-148b 0.93 9.94696E−06 0.757 miRNA 32 miR-483-5p −1.10  1.1191E−05 0.756 miRNA 48 ACA58_x 0.62 1.17327E−05 0.766 HAcaBox 89 miR-1260 0.94 1.21296E−05 0.776 miRNA 120 U108_x −0.50 1.23481E−05 0.763 HAcaBox 87 ACA61 −0.60 1.27104E−05 0.839 HAcaBox 135 14q-0 −0.58 1.39356E−05 0.767 CDBox 49 ACA16_x 0.55 1.40972E−05 0.763 HAcaBox 98 miR-30b-star 1.22 1.61096E−05 0.755 miRNA 61 ENSG00000201848_x −0.58 2.11059E−05 0.740 snoRNA 40 U3-2_s −0.57 2.19611E−05 0.800 CDBox 135 spike_in-control-2 −0.31 2.25868E−05 0.747 Oligonucleotide 137 spike-in controls miR-933 −0.56 2.89505E−05 0.776 miRNA 100 miR-181d 1.03 2.90988E−05 0.750 miRNA 61 HBII-239 0.45 2.92435E−05 0.744 CDBox 135 miR-497 1.17 2.92719E−05 0.730 miRNA 104 HBII-251 −0.48 3.04823E−05 0.797 CDBox 135 U51 −0.73 3.10371E−05 0.809 CDBox 134 miR-211 1.45 3.41149E−05 0.759 miRNA 84 miR-29a 1.10 3.68833E−05 0.777 miRNA 128 ENSG00000212538 −0.54 3.71864E−05 0.746 snoRNA 35 miR-886-3p 1.11 4.28603E−05 0.711 miRNA 110 miR-361-5p 0.58 4.51572E−05 0.859 miRNA 135 U51_x −0.48  4.6523E−05 0.773 CDBox 130 U46 0.59 4.67792E−05 0.744 CDBox 133 HBII-85-11 0.73 4.72933E−05 0.743 CDBox 31 ENSG00000207410_x −0.51 4.76931E−05 0.731 snoRNA 55 ENSG00000200492 −0.51 5.13688E−05 0.756 snoRNA 60 miR-503 1.05 5.22927E−05 0.730 miRNA 41 HBII-85-21_x 0.68 5.24419E−05 0.749 CDBox 39 miR-506 1.27 5.49222E−05 0.750 miRNA 76 ENSG00000212206_x −0.55 5.68573E−05 0.730 snoRNA 74 U64 0.49  5.7913E−05 0.755 HAcaBox 101 miR-629-star 1.07 5.89793E−05 0.745 miRNA 46 ENSG00000212284 −0.62 6.56826E−05 0.740 snoRNA 89 ENSG00000212423_x −0.60 6.59628E−05 0.728 snoRNA 115 U14B_x 0.58 6.71139E−05 0.714 CDBox 53 miR-324-3p 0.78 6.71385E−05 0.766 miRNA 100 miR-1287 0.79 7.37546E−05 0.733 miRNA 40 ENSG00000200897 −0.49 7.55587E−05 0.730 snoRNA 38 miR-498 −0.68 8.15427E−05 0.730 miRNA 56 U80 0.53 8.25012E−05 0.750 CDBox 132 14ql-8_x −0.55 8.60271E−05 0.740 CDBox 74 miR-181a 0.82 8.99722E−05 0.766 miRNA 134 HBII-166 −0.46 9.07119E−05 0.782 CDBox 134 miR-181a-star 1.03 9.30754E−05 0.727 miRNA 35 U17b 0.85 9.69173E−05 0.854 HAcaBox 135 U55 0.61 0.000102241 0.749 CDBox 135 ENSG00000207407 −0.43 0.000115453 0.706 snoRNA 56 ENSG00000212206 −0.50 0.000123184 0.701 snoRNA 46 miR-489 0.86 0.000125451 0.724 miRNA 58 hsa-let-7g 1.09 0.000128044 0.820 miRNA 127 ENSG00000199363 0.63 0.000135904 0.722 snoRNA 42 miR-27a 0.72 0.000140934 0.687 miRNA 136 ACA32 −0.40 0.000144838 0.746 HAcaBox 135 ENSG00000207022 −0.51 0.000149628 0.733 snoRNA 36 miR-371-5p −0.86 0.000152576 0.706 miRNA 64 miR-559 −0.73 0.000161415 0.728 miRNA 72 miR-193a-5p 0.92 0.000163281 0.777 miRNA 121 U23 0.46 0.00016459 0.724 HAcaBox 127 miR-196a-star −0.69 0.000167433 0.695 miRNA 37 U52 0.47 0.00016782 0.770 CDBox 135 ACA38 0.50 0.000170509 0.713 HAcaBox 42 miR-30e-star 0.82 0.000173654 0.736 miRNA 35 U49A_x −0.61 0.000183863 0.785 CDBox 135 U49B_x −0.54 0.000189603 0.729 CDBox 122 U71a 0.50 0.000191925 0.721 HAcaBox 50 HBII-95 0.42 0.000194646 0.724 CDBox 78 ENSG00000212587 −0.55 0.000201583 0.711 snoRNA 45 ENSG00000212401 −0.45 0.000208666 0.714 snoRNA 41 HBII-296A −0.45 0.000214033 0.713 CDBox 78 ACA67_x 0.49 0.000214513 0.730 HAcaBox 54 miR-494 0.79 0.000221314 0.725 miRNA 135 miR-17 0.77 0.00024803 0.782 miRNA 134 HBII-85-4_x −0.49 0.000264241 0.714 CDBox 125 miR-16 0.67 0.000270072 0.819 miRNA 135 miR-135b-star −0.68 0.000271929 0.710 miRNA 38 ACA37_x 0.47 0.00028645 0.718 HAcaBox 84 miR-126 0.82 0.000326027 0.780 miRNA 133 miR-200b 0.93 0.000339327 0.714 miRNA 109 miR-320d 0.89 0.000373646 0.811 miRNA 134 U100 0.41 0.000374728 0.702 scaRna 80 HBII-85-27_x −0.40 0.000383642 0.706 CDBox 56 HBII-85-15_x 0.60 0.000397454 0.706 CDBox 49 miR-885-3p −1.11 0.000407264 0.692 miRNA 73 miR-23b-star 0.76 0.000423736 0.723 miRNA 52 ENSG00000206909_x −0.46 0.000424153 0.710 snoRNA 47 U92 −0.50 0.000428521 0.750 scaRna 129 miR-628-3p 0.86 0.000458761 0.702 miRNA 38 miR-27b 0.70 0.00047114 0.718 miRNA 133 U88 −0.43 0.000500023 0.708 scaRna 69 miR-130a 1.00 0.000518644 0.729 miRNA 122 ACA4 0.42 0.00055437 0.673 HAcaBox 117 miR-151-5p 0.60 0.00055594 0.790 miRNA 135 ENSG00000200394 0.46 0.000561813 0.692 snoRNA 119 ACA5 0.48 0.000585113 0.708 HAcaBox 85 miR-1285 −0.74 0.000607741 0.698 miRNA 54 miR-664-star −0.82 0.000652475 0.686 miRNA 96 U56 −0.57 0.000672557 0.787 CDBox 135 ENSG00000200307 −0.44 0.000679263 0.686 snoRNA 86 miR-18b 0.81 0.000708619 0.688 miRNA 32 U76 0.38 0.000723275 0.712 CDBox 135 miR-378 0.75 0.000726542 0.769 miRNA 131 ENSG00000200307_x −0.43 0.000739867 0.716 snoRNA 50 miR-513b 1.02 0.000775353 0.701 miRNA 64 U56_x 0.54 0.000779671 0.716 CDBox 134 miR-642 −0.59 0.000808799 0.695 miRNA 69 miR-106a 0.66 0.000811882 0.788 miRNA 135 miR-379 0.94 0.000829873 0.702 miRNA 57 miR-570 −0.61 0.000842848 0.697 miRNA 88 ACA3-2 0.46 0.000855145 0.720 HAcaBox 135 U55_x 0.50 0.000899835 0.746 CDBox 135 ACA7_s −0.34 0.000995798 0.703 HAcaBox 133 miR-1228 −0.67 0.001047707 0.730 miRNA 114 miR-199a-3p 0.75 0.001282519 0.729 miRNA 134 ENSG00000212508 −0.59 0.001303596 0.687 snoRNA 122 ENSG00000200961 −0.41 0.001335597 0.699 snoRNA 41 miR-23b 0.45 0.00142683 0.817 miRNA 137 ENSG00000207177 −0.34 0.001464718 0.697 snoRNA 59 ACA50 0.41 0.001642667 0.674 HAcaBox 88 miR-550-star 0.75 0.001687411 0.692 miRNA 58 miR-617 −0.56 0.001827038 0.676 miRNA 64 U75 −0.57 0.001850734 0.729 CDBox 134 HBII-85-2_x −0.41 0.001956673 0.706 CDBox 135 miR-508-5p 0.77 0.001978936 0.711 miRNA 92 miR-200a 0.66 0.002095909 0.676 miRNA 33 miR-574-5p 0.79 0.002104165 0.749 miRNA 103 ACA49 −0.37 0.00221029 0.692 HAcaBox 127 miR-1826 0.46 0.002224183 0.799 miRNA 137 ENSG00000212377 −0.45 0.002249473 0.676 snoRNA 31 U45A −0.44 0.002283826 0.686 CDBox 74 miR-34a-star 0.61 0.002356105 0.682 miRNA 39 miR-378-star 0.71 0.002495647 0.710 miRNA 82 miR-509-5p 0.98 0.002612774 0.705 miRNA 81 miR-34a 0.79 0.00270219 0.676 miRNA 132 ACA2a 0.41 0.002741773 0.690 HAcaBox 55 miR-98 0.63 0.002795533 0.698 miRNA 47 miR-337-3p −0.59 0.002924904 0.709 miRNA 94 U58A −0.37 0.002957231 0.667 CDBox 129 miR-30e 0.66 0.003008812 0.673 miRNA 57 miR-196a 0.99 0.003061353 0.686 miRNA 77 miR-17-star 0.87 0.003088295 0.675 miRNA 56 HBII-429 −0.26 0.003238494 0.681 CDBox 137 HBII-85-5_x 0.38 0.003305314 0.678 CDBox 31 miR-191-star −0.50 0.003313493 0.701 miRNA 54 U102 −0.32 0.003411715 0.662 CDBox 124 ACA57 −0.35 0.003594842 0.737 scaRna 135 HBII-234_x −0.33 0.003636016 0.676 CDBox 102 hsa-let-7i 0.62 0.003881627 0.748 miRNA 135 spike_in-control-23 −0.30 0.003884651 0.673 Oligonucleotide 137 spike-in controls HBII-276 0.37 0.003895578 0.676 CDBox 132 mgU6-53 −0.37 0.004017934 0.667 CDBox 94 miR-575 −0.55 0.004110263 0.666 miRNA 41 miR-486-5p 1.06 0.004302169 0.672 miRNA 65 HBII-85-20_x 0.37 0.004413582 0.662 CDBox 42 U43 0.49 0.004416752 0.847 CDBox 135 miR-199b-3p 0.64 0.00460804 0.711 miRNA 134 U50B −0.47 0.004646908 0.678 CDBox 135 HBII-202 −0.37 0.004720422 0.711 CDBox 135 U32A_x 0.30 0.004742469 0.634 CDBox 136 U28_x 0.43 0.005077583 0.733 CDBox 133 miR-223 0.54 0.005512872 0.635 miRNA 83 U36A 0.26 0.005677159 0.654 CDBox 132 miR-548a-3p −0.65 0.005852929 0.669 miRNA 65 U82 −0.43 0.005931718 0.686 CDBox 131 ACA13 −0.40 0.005934282 0.697 HAcaBox 134 ENSG00000208308_x 0.48 0.005982879 0.755 snoRNA 129 14ql-8 −0.41 0.006020932 0.647 CDBox 61 ACA41_x 0.38 0.006458897 0.687 HAcaBox 122 U20 0.34 0.006504359 0.665 CDBox 117 HBII-210 −0.33 0.006967845 0.684 CDBox 135 ACA53 0.33 0.007009476 0.656 HAcaBox 105 miR-188-5p 0.59 0.00730559 0.668 miRNA 51 miR-653 −0.48 0.007396776 0.652 miRNA 50 ACA18_x 0.33 0.007506763 0.656 HAcaBox 135 ENSG00000202440_x −0.29 0.007646916 0.654 snoRNA 91 HBII-180C_x 0.33 0.007676115 0.666 CDBox 131 ENSG00000200235_x −0.29 0.007854884 0.650 snoRNA 59 HBII-85-17_x 0.43 0.008991412 0.650 CDBox 57 miR-205 0.39 0.009048012 0.665 miRNA 135 ACA27_x 0.24 0.009084132 0.676 HAcaBox 130 ACA52 0.31 0.009338308 0.664 HAcaBox 121 U8_x 0.37 0.00950871 0.638 CDBox 135 ENSG00000201229 0.35 0.009710558 0.662 snoRNA 32 miR-423-5p −0.49 0.009884603 0.704 miRNA 132 U72_x 0.27 0.009902439 0.642 HAcaBox 103 U50 0.37 0.010130893 0.679 CDBox 134 miR-34b 0.95 0.010634429 0.647 miRNA 32 U60 0.33 0.010937468 0.653 CDBox 103 ACA26 −0.38 0.011152582 0.688 scaRna 125 ACA21 −0.44 0.011353671 0.661 HAcaBox 133 U47 0.30 0.01143234 0.651 CDBox 74 miR-26a 0.40 0.011704306 0.793 miRNA 136 miR-21-star 0.54 0.012900809 0.648 miRNA 64 U43_x 0.42 0.013355042 0.808 CDBox 135 HBII-55 0.27 0.013604568 0.636 CDBox 135 ACA19 −0.29 0.013694584 0.634 HAcaBox 118 ENSG00000200897_x −0.34 0.013964134 0.628 snoRNA 70 ENSG00000207118 0.42 0.01420542 0.652 snoRNA 93 U31 −0.42 0.014421405 0.684 CDBox 132 miR-204 0.70 0.015040931 0.620 miRNA 53 miR-195 0.59 0.01545596 0.646 miRNA 132 miR-432 0.70 0.015655418 0.649 miRNA 51 hsa-let-7c 0.48 0.015958151 0.801 miRNA 135 ACA10_s 0.33 0.015973764 0.658 HAcaBox 113 miR-1825 −0.60 0.016002367 0.679 miRNA 132 14qll-1 −0.53 0.016171959 0.621 CDBox 128 14qll-21_x 0.32 0.017134601 0.670 CDBox 51 U34 0.24 0.017406393 0.609 CDBox 137 miR-138-1-star 0.64 0.017790103 0.611 miRNA 91 miR-1257 −0.40 0.018052873 0.633 miRNA 31 ACA51_x −0.23 0.018226343 0.656 HAcaBox 135 14qll-1_x −0.50 0.018262482 0.626 CDBox 131 ACA43 0.31 0.018272258 0.620 HAcaBox 114 hsa-let-7d 0.38 0.01921824 0.855 miRNA 135 14qll-12_x 0.40 0.020581734 0.652 CDBox 85 14qll-28_x 0.34 0.02070105 0.641 CDBox 51 spike_in-control-30 −0.36 0.02072199 0.657 Oligonucleotide 53 spike-in controls U49B_s −0.38 0.021350114 0.648 CDBox 129 U45B_x −0.32 0.022203975 0.615 CDBox 46 miR-125b 0.42 0.022489574 0.718 miRNA 135 ENSG00000202252 −0.23 0.023928736 0.654 snoRNA 135 miR-1324 −0.37 0.024126574 0.638 miRNA 57 HBI-61 −0.34 0.024172419 0.639 HAcaBox 134 miR-99b-star 0.34 0.024194238 0.626 miRNA 74 miR-20a 0.51 0.024441924 0.640 miRNA 134 U54 0.36 0.02454759 0.749 CDBox 135 ENSG00000200932 −0.31 0.024839208 0.656 snoRNA 47 ENSG00000212326 0.34 0.024965859 0.611 snoRNA 104 U95 −0.35 0.026080784 0.723 CDBox 135 miR-193b-star 0.57 0.028801534 0.684 miRNA 77 ACA5_x 0.29 0.0293861 0.662 HAcaBox 103 ACA1_x −0.30 0.029505966 0.627 HAcaBox 51 mgU6-53_x −0.26 0.03076806 0.618 CDBox 113 U26 −0.36 0.030778322 0.608 CDBox 135 miR-24 0.36 0.031162316 0.771 miRNA 135 spike_in-control-29 −0.24 0.03150161 0.615 Oligonucleotide 137 spike-in controls miR-19b 0.57 0.03221182 0.612 miRNA 124 miR-513c 0.78 0.032937767 0.632 miRNA 73 U42B_x 0.27 0.033688384 0.613 CDBox 109 miR-20a-star −0.90 0.033742183 0.639 miRNA 61 U25 −0.30 0.035318167 0.655 CDBox 135 miR-455-3p 0.34 0.036186611 0.643 miRNA 136 U109 0.32 0.036189693 0.634 scaRna 69 miR-938 −0.32 0.037572276 0.639 miRNA 45 ENSG00000207503 −0.27 0.037791965 0.622 snoRNA 42 HBII-289 −0.24 0.041306388 0.652 CDBox 135 U17b_x 0.34 0.041402099 0.728 HAcaBox 135 ACA8_x −0.28 0.042136422 0.624 HAcaBox 125 ACA44 0.29 0.043557966 0.693 HAcaBox 135 U45C_x −0.33 0.043838427 0.586 CDBox 72 ENSG00000201133 −0.27 0.045035669 0.645 snoRNA 35 ACA55 −0.26 0.045152107 0.606 HAcaBox 85 miR-451 0.53 0.046730942 0.628 miRNA 72 14qll-26 −0.32 0.047726134 0.619 CDBox 32 miR-141 0.44 0.049534195 0.627 miRNA 69

TABLE 2 50 miRNAs most significantly expressed between melanoma and nevi Probe Name Log2-FC P value AUC ProbeType #Sample Detected miR-132 2.98  1.2728E−32 0.994 miRNA 96 miR-150 3.27 5.83192E−30 1.000 miRNA 117 miR-339-5p 2.73 1.69388E−27 0.988 miRNA 75 miR-15b 3.02 6.41784E−27 0.978 miRNA 125 miR-342-3p 2.23 7.03798E−26 0.993 miRNA 136 miR-572 −2.78 1.00008E−25 0.970 miRNA 116 miR-155 4.14 1.28454E−25 0.981 miRNA 123 miR-425 2.74 8.13135E−25 0.975 miRNA 114 miR-1202 −2.63 2.97795E−23 0.986 miRNA 57 miR-1268 −2.68 6.05905E−23 0.997 miRNA 133 HBII-382_s −1.71 6.58931E−22 0.971 scaRna 126 miR-1225-5p −2.36 1.05361E−21 0.953 miRNA 90 miR-30c 2.39 2.58594E−21 0.980 miRNA 125 miR-106b-star 2.24 3.48507E−21 0.961 miRNA 72 miR-125a-5p 2.39 6.90609E−21 0.967 miRNA 128 mgU6-53B −1.51 1.74782E−20 0.973 CDBox 99 miR-25 2.57 1.52769E−19 0.956 miRNA 118 miR-149-star −2.03 1.64858E−19 1.000 miRNA 135 miR-939 −2.21  1.9129E−19 0.973 miRNA 57 miR-92b-star −2.30 2.05085E−19 0.969 miRNA 111 miR-500-star 2.38 3.11256E−19 0.956 miRNA 97 miR-22 2.69  3.8844E−19 0.965 miRNA 120 HBII-142_x −1.22 4.93318E−19 0.981 CDBox 135 miR-181b 2.41 1.00639E−18 0.960 miRNA 130 HBII-142 −1.33 1.18794E−18 0.988 CDBox 135 U38B −1.95 1.35964E−18 0.975 CDBox 134 miR-663 −2.10 2.04672E−18 0.984 miRNA 134 miR-1224-5p −2.54 2.91169E−18 0.946 miRNA 78 miR-23a 1.18 2.99523E−18 0.967 miRNA 137 HBII-85-6_x −1.73 4.78418E−18 0.939 CDBox 137 miR-1207-5p −2.14 4.98991E−18 0.995 miRNA 133 miR-1301 2.36 5.22389E−18 0.931 miRNA 54 miR-1228-star −2.41 5.51513E−18 0.997 miRNA 134 miR-345 2.45 6.43552E−18 0.942 miRNA 75 miR-30a-star 2.32 6.92165E−18 0.932 miRNA 64 ENSG00000199411_s −1.97 7.23325E−18 0.991 snoRNA 135 ENSG00000202327 −1.40 8.75839E−18 0.938 snoRNA 54 miR-92a 1.67 9.49427E−18 0.979 miRNA 136 miR-127-3p 2.43 9.84615E−18 0.936 miRNA 89 HBII-85-26 −2.07 1.50619E−17 0.951 CDBox 136 miR-1308 −2.14 1.99888E−17 0.999 miRNA 135 miR-31 3.29 2.46697E−17 0.913 miRNA 103 miR-921 −1.46 2.61089E−17 0.928 miRNA 49 miR-146b-5p 2.37 6.10422E−17 0.918 miRNA 83 miR-768-3p −1.05 6.66153E−17 0.949 miRNA 137 miR-708 2.29 6.86187E−17 0.929 miRNA 102 miR-139-5p 2.23 2.78962E−16 0.922 miRNA 80 ACA24_x 1.33 3.37973E−16 0.925 HAcaBox 98 miR-501-3p 1.83 4.76751E−16 0.923 miRNA 80 miR-502-3p 2.16 5.19509E−16 0.925 miRNA 110 miR-923 −1.86 5.55191E−16 0.996 miRNA 135

TABLE 3 miRNAs significantly expressed between melanoma and normal skin Log2- #Sample Probe Name FC P value AUC ProbeType Detected miR-146a 4.6 9.43624E−20 0.983 miRNA 129 miR-509-3p 5.2 5.61558E−17 0.969 miRNA 116 14qll-14 −2.6 1.16829E−14 0.978 CDBox 56 miR-25 2.4   2.08E−14 0.949 miRNA 118 miR-138 3.2 2.51219E−13 0.954 miRNA 82 miR-509-3-5p 3.6 2.94532E−13 0.963 miRNA 107 miR-506 3.4 6.99753E−13 0.954 miRNA 76 14qll-14_x −2.2 8.36026E−13 0.970 CDBox 56 14ql-4 −2.1  8.9267E−13 0.964 CDBox 80 miR-30b 2.2 2.43822E−12 0.957 miRNA 128 miR-513a-5p 3.5 3.17158E−12 0.939 miRNA 105 miR-21 3.3 4.58391E−12 0.926 miRNA 79 Z17B −1.0 6.92137E−12 0.956 CDBox 117 U33 −0.9 8.19977E−12 0.968 CDBox 137 miR-20b 2.8 2.24496E−11 0.939 miRNA 99 hsa-let-7i 2.3 3.27742E−11 0.973 miRNA 135 HBII-239 −1.0  3.9295E−11 0.920 CDBox 135 miR-146b-5p 2.3 8.71488E−11 0.910 miRNA 83 14qll-26_x −1.8 9.17329E−11 0.914 CDBox 41 miR-155 2.8 1.12361E−10 0.940 miRNA 123 miR-151-3p 1.8  1.7141E−10 0.922 miRNA 117 HBII-289 −1.1 1.76712E−10 0.963 CDBox 135 14qll-12_x −1.7  1.9475E−10 0.907 CDBox 85 miR-1274a 1.9 3.28343E−10 0.928 miRNA 48 HBII-180A_x −0.8 4.68853E−10 0.933 CDBox 123 miR-1301 2.0 7.22101E−10 0.896 miRNA 54 14qll-1_x −1.7 7.72545E−10 0.965 CDBox 131 miR-193b −1.4  8.7852E−10 0.974 miRNA 130 miR-510 2.7 1.03096E−09 0.914 miRNA 82 miR-126 2.1 1.35297E−09 0.959 miRNA 133 miR-24-2-star 1.8 1.36986E−09 0.895 miRNA 63 miR-106b 2.1 1.91606E−09 0.930 miRNA 127 HBII-276 −1.0 2.06051E−09 0.908 CDBox 132 miR-532-5p 1.8  2.9654E−09 0.887 miRNA 113 14qll-12 −1.7 3.71443E−09 0.859 CDBox 58 miR-19b 2.1 3.71682E−09 0.908 miRNA 124 miR-30a 1.6 6.61873E−09 0.894 miRNA 113 HBII-85-26_x −1.1 7.76845E−09 0.895 CDBox 135 miR-150 1.7 1.08172E−08 0.894 miRNA 117 14qll-26 −1.5 1.16704E−08 0.869 CDBox 32 miR-324-5p 1.6 1.24724E−08 0.880 miRNA 102 14ql-8 −1.2 1.39067E−08 0.894 CDBox 61 miR-185 1.6 1.44139E−08 0.952 miRNA 132 miR-194 1.8 1.72961E−08 0.883 miRNA 54 14qll-1 −1.5 2.76507E−08 0.928 CDBox 128 HBII-202 −1.0 2.89826E−08 0.946 CDBox 135 miR-768-5p −1.6  3.1365E−08 0.986 miRNA 135 miR-421 1.6 4.42916E−08 0.872 miRNA 68 miR-28-5p 1.7 5.45027E−08 0.877 miRNA 105 miR-151-5p 1.2 5.48542E−08 0.968 miRNA 135 miR-15a 2.0 5.77662E−08 0.889 miRNA 80 miR-26a 1.3  6.8712E−08 0.968 miRNA 136 U25 −1.0 7.35643E−08 0.976 CDBox 135 HBII-180C −0.9 7.47464E−08 0.836 CDBox 115 miR-584 1.9  8.0812E−08 0.863 miRNA 57 hsa-let-7f 2.2 1.05873E−07 0.924 miRNA 130 miR-1268 −1.7 1.09503E−07 0.954 miRNA 133 miR-572 −1.4 1.12523E−07 0.886 miRNA 116 ENSG00000200897 −0.9 1.38587E−07 0.843 snoRNA 38 miR-508-5p 1.8 1.57345E−07 0.873 miRNA 92 hsa-let-7g 1.8 1.57818E−07 0.916 miRNA 127 miR-20a 1.7 1.58351E−07 0.926 miRNA 134 miR-1225-5p −1.3  6.2291E−07 0.890 miRNA 90 U55 −0.8  1.8721E−07 0.942 CDBox 135 miR-509-5p 2.3 1.95354E−07 0.864 miRNA 81 HBII-85-6_x −1.1  2.0107E−07 0.872 CDBox 137 U99 −0.7 2.22915E−07 0.872 HAcaBox 137 miR-501-3p 1.4 2.27557E−07 0.858 miRNA 80 miR-29a 1.8 2.42514E−07 0.907 miRNA 128 miR-1274b 1.6  2.6096E−07 0.863 miRNA 112 miR-210 −1.2 2.81748E−07 0.895 miRNA 130 HBII-85-26 −1.4  3.5949E−07 0.896 CDBox 136 miR-199a-5p −0.8 3.93185E−07 0.870 miRNA 129 miR-149 −1.6 5.63545E−07 0.920 miRNA 100 miR-1307 −0.9  5.6459E−07 0.907 miRNA 97 miR-744 −1.1 5.78243E−07 0.971 miRNA 133 miR-92b-star −1.4 6.45948E−07 0.907 miRNA 111 ACA11 0.8 7.08497E−07 0.842 HAcaBox 48 miR-27a 1.0 7.17204E−07 0.887 miRNA 136 miR-34a-star 1.4 8.89848E−07 0.842 miRNA 39 U46 −0.8  9.2248E−07 0.884 CDBox 133 miR-214 −1.2 1.02234E−06 0.930 miRNA 135 miR-132 1.2 1.15319E−06 0.808 miRNA 96 miR-18a 2.0  1.3554E−06 0.866 miRNA 77 miR-125a-5p −1.0 1.86884E−06 0.934 miRNA 128 14ql-8_x −0.9 1.95428E−06 0.831 CDBox 74 miR-663 −1.3  1.9913E−06 0.910 miRNA 134 miR-16 1.1 2.78721E−06 0.955 miRNA 135 HBII-142_x −0.8 2.95334E−06 0.909 CDBox 135 U103_s −0.8  3.6466E−06 0.825 CDBox 111 U55_x −0.8 3.84586E−06 0.958 CDBox 135 HBII-142 −0.8 3.88875E−06 0.917 CDBox 135 miR-130b 1.9 4.19812E−06 0.858 miRNA 90 miR-339-3p 1.1 4.25751E−06 0.796 miRNA 60 miR-30d 1.2 4.40714E−06 0.902 miRNA 131 miR-196a 2.0 4.53608E−06 0.833 miRNA 77 miR-199a-3p 1.4 5.28457E−06 0.889 miRNA 134 miR-211 2.1  5.7974E−06 0.859 miRNA 84 miR-30e 1.2 5.88414E−06 0.839 miRNA 57 ACA54 −0.6 7.09713E−06 0.861 HAcaBox 133 miR-106b-star 1.1 7.86756E−06 0.720 miRNA 72 14qll-3 −1.1 8.39354E−06 0.832 CDBox 95 HBII-55 −0.6 8.41728E−06 0.860 CDBox 135 miR-486-5p −2.2 1.02088E−05 0.829 miRNA 65 miR-145 −1.3 1.03604E−05 0.831 miRNA 135 miR-34a 1.4 1.04763E−05 0.868 miRNA 132 miR-106a 1.2 1.06568E−05 0.919 miRNA 135 miR-1271 1.3 1.06608E−05 0.829 miRNA 70 miR-500 1.3 1.07278E−05 0.816 miRNA 86 miR-625 1.4 1.08708E−05 0.800 miRNA 59 14qll-9_x −0.8 1.14849E−05 0.821 CDBox 36 14qll-21_x −0.9 1.17647E−05 0.797 CDBox 51 U83 −0.6 1.19481E−05 0.889 CDBox 135 miR-638 −1.3 1.34963E−05 0.914 miRNA 135 HBII-180C_x −0.6 1.39632E−05 0.841 CDBox 131 ENSG00000212139_x −0.6 1.67825E−05 0.831 snoRNA 124 miR-628-3p 1.4 1.76296E−05 0.811 miRNA 38 U104 −0.8 1.84466E−05 0.886 CDBox 135 miR-10b 1.2 2.02886E−05 0.829 miRNA 106 U34 −0.4 2.06445E−05 0.807 CDBox 137 miR-127-3p −1.1 2.16929E−05 0.799 miRNA 89 14qll-28_x −0.9 2.21554E−05 0.815 CDBox 51 miR-1207-5p −1.2 2.25272E−05 0.917 miRNA 133 miR-199b-3p 1.3 2.29577E−05 0.863 miRNA 134 U35A −0.7 2.50745E−05 0.900 CDBox 135 U91_s −0.8 2.92768E−05 0.806 scaRna 132 spike_in-control-36 0.6 2.95775E−05 0.865 Oligonucleotide 137 spike-in controls U32A_x −0.4 3.05381E−05 0.827 CDBox 136 miR-21-star 1.2 3.24139E−05 0.808 miRNA 64 miR-149-star −1.1 4.06943E−05 0.888 miRNA 135 miR-193b-star −1.4 4.09065E−05 0.831 miRNA 77 U67_x 0.8 4.09255E−05 0.792 HAcaBox 50 ACA20 −0.8 4.42355E−05 0.839 HAcaBox 131 miR-1287 1.2 4.69003E−05 0.775 miRNA 40 ACA64 −0.7 4.84198E−05 0.802 HAcaBox 33 U57 −0.8 5.04018E−05 0.944 CDBox 135 miR-193a-3p 1.1 5.08442E−05 0.766 miRNA 42 miR-371-5p 1.1 5.19682E−05 0.775 miRNA 64 HBII-85-8_x −0.7 5.46471E−05 0.782 CDBox 135 miR-503 1.4  5.4991E−05 0.794 miRNA 41 miR-138-1-star 1.5 5.60721E−05 0.877 miRNA 91 ACA67_x 0.7 5.78125E−05 0.790 HAcaBox 54 miR-1228-star −1.3 5.81565E−05 0.895 miRNA 134 U41 −0.8 5.85491E−05 0.875 CDBox 135 miR-193a-5p −1.2 5.98826E−05 0.897 miRNA 121 ENSG00000202252 −0.5 6.64141E−05 0.839 snoRNA 135 14ql-4_x −1.0  6.7457E−05 0.760 CDBox 51 miR-508-3p 1.7 6.91432E−05 0.801 miRNA 74 miR-200b 1.4 7.07093E−05 0.779 miRNA 109 miR-660 1.2 7.13979E−05 0.791 miRNA 71 U67 0.8 7.16134E−05 0.774 HAcaBox 44 U50B −0.8  7.249E−05 0.869 CDBox 135 miR-513c 1.9 7.46404E−05 0.771 miRNA 73 14ql-9_x −0.7 7.67649E−05 0.772 CDBox 34 U60 −0.6 8.34902E−05 0.779 CDBox 103 U102 0.5 8.47602E−05 0.772 CDBox 124 EN8G00000200235_x −0.5  9.4306E−05 0.793 snoRNA 59 miR-17 1.0 9.79941E−05 0.910 miRNA 134 U36C −0.6 0.000107866 0.854 CDBox 135 miR-491-5p 0.9 0.000121664 0.753 miRNA 92 miR-425 1.0 0.000138461 0.771 miRNA 114 miR-500-star 1.0 0.000148221 0.761 miRNA 97 U78_x 1.0 0.000150233 0.872 CDBox 134 ENSG00000202327 −0.6 0.000153478 0.783 snoRNA 54 miR-181c 1.1 0.000156102 0.772 miRNA 59 ENSG00000212523_x −0.8 0.000165203 0.772 snoRNA 135 mgU6-53B_x −0.5 0.00016649 0.784 CDBox 121 miR-99b-star 0.8 0.00017311 0.743 miRNA 74 U53 −0.7 0.000175804 0.763 CDBox 126 miR-296-3p −1.0 0.000180884 0.771 miRNA 90 U50B_x −0.7 0.000181435 0.829 CDBox 135 miR-30a-star 1.2 0.00018331 0.766 miRNA 64 U46_x −0.8 0.000183742 0.819 CDBox 131 ENSG00000207410_x −0.5 0.000198904 0.779 snoRNA 55 miR-99a −0.8 0.000204711 0.829 miRNA 135 miR-513b 1.4 0.000214575 0.781 miRNA 64 miR-23a 0.6 0.000218135 0.908 miRNA 137 miR-31 1.7 0.00023269 0.721 miRNA 103 miR-345 1.2 0.000250483 0.782 miRNA 75 miR-212 1.5 0.000254534 0.831 miRNA 62 ENSG00000212266 −0.6 0.000255339 0.777 snoRNA 93 miR-152 1.1 0.000260732 0.813 miRNA 122 U19 0.6 0.000277432 0.757 HAcaBox 118 miR-886-3p 1.0 0.000298839 0.768 miRNA 110 U73a −0.6 0.000338569 0.846 CDBox 135 miR-423-3p −0.7 0.000347496 0.848 miRNA 107 mgh28S-2411 −0.6 0.00034785 0.901 CDBox 135 miR-103 0.8 0.000430411 0.930 miRNA 135 miR-629 1.1 0.000473215 0.769 miRNA 64 U65 −0.6 0.000562655 0.778 HAcaBox 128 14ql-7 −0.7 0.000569692 0.803 CDBox 51 miR-933 −0.6 0.000572446 0.769 miRNA 100 miR-205 −0.6 0.000653671 0.767 miRNA 135 U105 −0.4 0.000692027 0.749 CDBox 126 miR-378-star 1.1 0.000731339 0.769 miRNA 82 miR-939 −0.9 0.000748523 0.822 miRNA 57 hsa-let-7d 0.8 0.000759497 0.978 miRNA 135 miR-362-5p 1.2 0.000775756 0.762 miRNA 94 ACA9 0.7 0.000834583 0.739 HAcaBox 82 miR-10a 1.1 0.000844868 0.759 miRNA 69 miR-877 −0.9 0.000845569 0.789 miRNA 126 miR-128 1.1 0.000865867 0.762 miRNA 44 miR-27b 0.8 0.000876954 0.799 miRNA 133 miR-17-star 1.2 0.000905238 0.748 miRNA 56 mgh18S-121 −0.5 0.000964406 0.789 CDBox 134 ENSG00000200394 0.5 0.000968073 0.772 snoRNA 119 ENSG00000212315 0.6 0.000971189 0.757 snoRNA 117 ENSG00000212615_x −0.5 0.000998331 0.707 snoRNA 102 miR-148a 0.9 0.001014157 0.733 miRNA 41 miR-923 −0.9 0.001032772 0.826 miRNA 135 ACA20_x −0.6 0.001087206 0.830 HAcaBox 134 ENSG00000212579_x −0.6 0.001126846 0.765 snoRNA 100 miR-589-star 0.5 0.001225281 0.703 miRNA 43 miR-107 0.7 0.001260426 0.932 miRNA 135 ENSG00000199363 0.6 0.001425983 0.750 snoRNA 42 ACA55 0.5 0.001430114 0.746 HAcaBox 85 U54 −0.7 0.001511402 0.868 CDBox 135 U36A −0.4 0.001519955 0.759 CDBox 132 U95 −0.7 0.001551616 0.929 CDBox 135 14qll-22_x −0.7 0.001591836 0.701 CDBox 50 U58B_x −0.5 0.001603166 0.726 CDBox 132 miR-720 0.7 0.001622509 0.760 miRNA 137 ENSG00000200394_x 0.5 0.001742151 0.711 snoRNA 115 HBII-166 −0.5 0.001874456 0.769 CDBox 134 miR-29b-1-star 1.3 0.001938943 0.736 miRNA 46 HBII-436 −0.5 0.001978078 0.731 CDBox 102 U23 −0.5 0.002014594 0.748 HAcaBox 127 miR-346 −0.9 0.002054981 0.774 miRNA 55 U58A −0.5 0.002095349 0.735 CDBox 129 HBII-95 0.5 0.00219824 0.703 CDBox 78 miR-339-5p 0.7 0.002357824 0.693 miRNA 75 ACA25 0.4 0.002440171 0.724 HAcaBox 73 miR-363 1.1 0.002716266 0.735 miRNA 44 miR-432 −1.1 0.002752773 0.738 miRNA 51 miR-424-star 1.1 0.002765097 0.718 miRNA 41 U27 −0.6 0.002765603 0.792 CDBox 134 miR-30c 0.6 0.002778453 0.770 miRNA 125 miR-93 0.7 0.002813996 0.863 miRNA 134 hsa-let-7a 0.8 0.002856983 0.911 miRNA 136 miR-181d 1.0 0.002954008 0.748 miRNA 61 miR-99b −0.5 0.00296227 0.757 miRNA 129 miR-550-star 0.9 0.003086147 0.740 miRNA 58 U43 −0.7 0.00327637 0.865 CDBox 135 U52 −0.5 0.003332494 0.783 CDBox 135 miR-502-3p 0.8 0.003628993 0.775 miRNA 110 spike_in-control-29 −0.4 0.003663934 0.723 Oligonucleotide 137 spike-in controls 14qll-17 −0.7 0.003689916 0.703 CDBox 71 ENSG00000212302_x −0.4 0.003876466 0.676 snoRNA 56 miR-574-3p −0.7 0.003955226 0.842 miRNA 122 miR-320d −0.8 0.004248663 0.868 miRNA 134 miR-1246 1.0 0.004275913 0.772 miRNA 131 ACA7_s −0.4 0.004475709 0.719 HAcaBox 133 miR-15b 0.6 0.004714973 0.792 miRNA 125 HBII-52-37_x −0.5 0.004773282 0.714 CDBox 37 gi555853_copy0 −0.6 0.004784885 0.799 5.8s rRNA 137 miR-30e-star 0.8 0.00500785 0.717 miRNA 35 miR-18a-star 0.8 0.005024206 0.723 miRNA 40 ENSG00000201847_x −0.4 0.005059414 0.688 snoRNA 48 gi555853_copy5 −0.5 0.005220274 0.809 5.8s rRNA 137 U24 −0.4 0.005634443 0.708 CDBox 133 miR-1280 −0.4 0.005689807 0.740 miRNA 134 miR-1231 −0.8 0.005810993 0.676 miRNA 79 ENSG00000202498_x −0.5 0.005898128 0.701 snoRNA 137 gi555853_copy7 −0.5 0.006325433 0.804 5.8s rRNA 137 miR-28-3p 0.9 0.006506887 0.711 miRNA 94 ENSG00000199435 0.4 0.006656491 0.706 snoRNA 63 gi555853_copy2 −0.5 0.00671481 0.800 5.8s rRNA 137 U43_x −0.6 0.006913554 0.854 CDBox 135 ENSG00000212273_x −0.5 0.007104981 0.725 snoRNA 126 ENSG00000200961 −0.4 0.007247826 0.716 snoRNA 41 U38A −0.7 0.007390068 0.814 CDBox 134 E3_x −0.4 0.007704052 0.779 HAcaBox 135 gi555853_copy8 −0.5 0.00776022 0.799 5.8s rRNA 137 HBII-234_x −0.3 0.007814833 0.703 CDBox 102 U28 −0.5 0.008026327 0.771 CDBox 135 ENSG00000212587 −0.5 0.008211191 0.685 snoRNA 45 ENSG00000212149_x −0.5 0.008233504 0.678 snoRNA 77 HBII-420 −0.5 0.00828299 0.771 CDBox 131 gi555853_copy1 −0.5 0.008395769 0.791 5.8s rRNA 137 HBII-85-21_x 0.5 0.008439987 0.717 CDBox 39 gi555853_copy4 −0.5 0.008541113 0.793 5.8s rRNA 137 gi555853_copy6 −0.5 0.008815947 0.792 5.8s rRNA 137 ENSG00000200897_x −0.5 0.009024011 0.686 snoRNA 70 ENSG00000207002 −0.4 0.009046282 0.706 snoRNA 35 ENSG00000212627 −0.4 0.009345896 0.714 snoRNA 69 gi555853_copy3 −0.5 0.009628403 0.777 5.8s rRNA 137 HBII-85-17_x 0.5 0.009677702 0.685 CDBox 57 miR-92b 0.5 0.009817789 0.672 miRNA 91 HBII-210 −0.4 0.009959111 0.756 CDBox 135 ACA3 −0.5 0.010166004 0.740 HAcaBox 134 miR-769-5p 0.7 0.010207709 0.622 miRNA 56 U100 0.4 0.010232137 0.645 scaRna 80 U13 −0.4 0.010247574 0.720 CDBox 135 miR-181b 0.6 0.010316868 0.816 miRNA 130 ACA46 0.4 0.010365242 0.699 HAcaBox 124 gi555853_copy9 −0.5 0.010460125 0.788 5.8s rRNA 137 U70_x −0.5 0.011265988 0.704 HAcaBox 53 HBII-85-15_x 0.5 0.011411104 0.695 CDBox 49 miR-203 0.7 0.01182754 0.822 miRNA 132 miR-320c −0.6 0.011901129 0.845 miRNA 135 miR-191-star −0.6 0.012197187 0.680 miRNA 54 ACA18_x −0.4 0.012258626 0.694 HAcaBox 135 miR-27a-star 0.8 0.013060406 0.702 miRNA 40 miR-192 0.7 0.013123545 0.703 miRNA 32 U74_x 0.5 0.013418084 0.804 CDBox 135 ACA15_x −0.3 0.013532632 0.691 HAcaBox 124 miR-198 −0.6 0.013839541 0.687 miRNA 31 U28_x −0.5 0.013863106 0.711 CDBox 133 ACA28 0.4 0.013886424 0.685 HAcaBox 120 U97 −0.4 0.013970203 0.692 CDBox 133 ACA4 0.3 0.014571497 0.655 HAcaBox 117 miR-423-5p −0.6 0.015447652 0.754 miRNA 132 ACA25_x 0.4 0.015584731 0.701 HAcaBox 114 HBII-382_s −0.4 0.016066802 0.670 scaRna 126 miR-34b 1.2 0.016083233 0.693 miRNA 32 miR-181a-2-star 0.7 0.016427521 0.646 miRNA 99 U15B −0.3 0.017584552 0.689 CDBox 129 miR-361-5p 0.4 0.017617483 0.783 miRNA 135 ACA9_x 0.5 0.017621407 0.669 HAcaBox 77 ACA15_s −0.3 0.017633335 0.738 HAcaBox 116 miR-23b-star 0.7 0.018210894 0.672 miRNA 52 ACA60 −0.3 0.018634054 0.702 HAcaBox 129 14q-0 −0.4 0.019666039 0.681 CDBox 49 HBII-85-22_x 0.5 0.019999179 0.673 CDBox 42 miR-30b-star 0.9 0.020172985 0.686 miRNA 61 miR-125b −0.5 0.020376572 0.707 miRNA 135 ENSG00000199411_s −0.6 0.020504379 0.779 snoRNA 135 HBII-251 −0.3 0.020538747 0.679 CDBox 135 miR-483-3p 0.9 0.020547545 0.666 miRNA 40 ACA23 −0.3 0.021199129 0.685 HAcaBox 113 ENSG00000212432_s −0.4 0.021742945 0.649 snoRNA 75 miR-25-star 0.7 0.022483461 0.671 miRNA 52 ENSG00000200932 −0.3 0.023007003 0.687 snoRNA 47 U49B_s 0.5 0.023157964 0.726 CDBox 129 ENSG00000212214_x 0.4 0.023504965 0.687 snoRNA 88 U83B −0.4 0.024471226 0.745 CDBox 135 miR-320b −0.6 0.025686249 0.790 miRNA 135 miR-489 0.6 0.025963923 0.680 miRNA 58 miR-26b 0.7 0.026201573 0.682 miRNA 45 U14B −0.4 0.027046414 0.632 CDBox 51 HBII-115 −0.3 0.027115569 0.730 CDBox 117 ENSG00000207027 −0.4 0.028391297 0.621 snoRNA 36 miR-494 0.5 0.029570695 0.689 miRNA 135 miR-181a-star 0.7 0.030787798 0.668 miRNA 35 U49A_x 0.4 0.03088655 0.750 CDBox 135 ACA14b_x −0.3 0.030912946 0.693 HAcaBox 105 U21 −0.4 0.031731894 0.691 CDBox 134 HBII-135_x 0.6 0.032076241 0.663 CDBox 132 miR-382 0.8 0.032245385 0.651 miRNA 47 miR-532-3p 0.4 0.032767036 0.697 miRNA 95 miR-214-star 0.8 0.032818193 0.670 miRNA 47 mgU6-53B −0.3 0.033293267 0.663 CDBox 99 miR-200c −0.7 0.033609817 0.659 miRNA 134 miR-575 0.5 0.034489882 0.657 miRNA 41 mgU6-53 −0.3 0.034658143 0.674 CDBox 94 miR-422a 0.8 0.035772116 0.674 miRNA 84 ENSG00000201848 −0.3 0.036190647 0.652 snoRNA 34 ACA45 0.3 0.036575475 0.653 scaRna 53 miR-559 −0.5 0.036810259 0.647 miRNA 72 ACA61 −0.4 0.037187144 0.716 HAcaBox 135 snR38C −0.4 0.037656608 0.721 CDBox 134 HBII-85-23_x 0.5 0.037660456 0.650 CDBox 58 miR-551b-star −0.5 0.037971736 0.645 miRNA 61 miR-27b-star 0.7 0.041289539 0.663 miRNA 51 U50 −0.4 0.041514005 0.696 CDBox 134 ACA58_x 0.3 0.041793061 0.653 HAcaBox 89 ACA53 −0.3 0.0421874 0.635 HAcaBox 105 U49A 0.4 0.042284609 0.694 CDBox 135 ACA16 −0.4 0.042968027 0.658 HAcaBox 47 ACA13 −0.4 0.043493352 0.722 HAcaBox 134 miR-320a −0.5 0.043683231 0.755 miRNA 135 ENSG00000200969 −0.4 0.043904585 0.663 snoRNA 67 miR-642 −0.4 0.04542452 0.640 miRNA 69 miR-148b 0.5 0.046811178 0.645 miRNA 32 U84 −0.2 0.046963896 0.683 CDBox 131 U56_x 0.4 0.047943262 0.701 CDBox 134 miR-451 0.7 0.049304242 0.632 miRNA 72 miR-195 0.6 0.049397144 0.698 miRNA 132

TABLE 4 50 miRNAs most significantly expressed between melanoma and normal skin Log2- #Sample Probe Name FC P value AUC ProbeType Detected miR-146a 4.6 9.43624E−20 0.983 miRNA 129 miR-509-3p 5.2 5.61558E−17 0.969 miRNA 116 14qll-14 −2.6 1.16829E−14 0.978 CDBox 56 miR-25 2.4   2.08E−14 0.949 miRNA 118 miR-138 3.2 2.51219E−13 0.954 miRNA 82 miR-509-3-5p 3.6 2.94532E−13 0.963 miRNA 107 miR-506 3.4 6.99753E−13 0.954 miRNA 76 14qll-14_x −2.2 8.36026E−13 0.970 CDBox 56 14ql-4 −2.1  8.9267E−13 0.964 CDBox 80 miR-30b 2.2 2.43822E−12 0.957 miRNA 128 miR-513a-5p 3.5 3.17158E−12 0.939 miRNA 105 miR-21 3.3 4.58391E−12 0.926 miRNA 79 Z17B −1.0 6.92137E−12 0.956 CDBox 117 U33 −0.9 8.19977E−12 0.968 CDBox 137 miR-20b 2.8 2.24496E−11 0.939 miRNA 99 hsa-let-7i 2.3 3.27742E−11 0.973 miRNA 135 HBII-239 −1.0  3.9295E−11 0.920 CDBox 135 miR-146b-5p 2.3 8.71488E−11 0.910 miRNA 83 14qll-26_x −1.8 9.17329E−11 0.914 CDBox 41 miR-155 2.8 1.12361E−10 0.940 miRNA 123 miR-151-3p 1.8  1.7141E−10 0.922 miRNA 117 HBII-289 −1.1 1.76712E−10 0.963 CDBox 135 14qll-12_x −1.7  1.9475E−10 0.907 CDBox 85 miR-1274a 1.9 3.28343E−10 0.928 miRNA 48 HBII-180A_x −0.8 4.68853E−10 0.933 CDBox 123 miR-1301 2.0 7.22101E−10 0.896 miRNA 54 14qll-1_x −1.7 7.72545E−10 0.965 CDBox 131 miR-193b −1.4  8.7852E−10 0.974 miRNA 130 miR-510 2.7 1.03096E−09 0.914 miRNA 82 miR-126 2.1 1.35297E−09 0.959 miRNA 133 miR-24-2-star 1.8 1.36986E−09 0.895 miRNA 63 miR-106b 2.1 1.91606E−09 0.930 miRNA 127 HBII-276 −1.0 2.06051E−09 0.908 CDBox 132 miR-532-5p 1.8  2.9654E−09 0.887 miRNA 113 14qll-12 −1.7 3.71443E−09 0.859 CDBox 58 miR-19b 2.1 3.71682E−09 0.908 miRNA 124 miR-30a 1.6 6.61873E−09 0.894 miRNA 113 HBII-85-26_x −1.1 7.76845E−09 0.895 CDBox 135 miR-150 1.7 1.08172E−08 0.894 miRNA 117 14qll-26 −1.5 1.16704E−08 0.869 CDBox 32 miR-324-5p 1.6 1.24724E−08 0.880 miRNA 102 14ql-8 −1.2 1.39067E−08 0.894 CDBox 61 miR-185 1.6 1.44139E−08 0.952 miRNA 132 miR-194 1.8 1.72961E−08 0.883 miRNA 54 14qll-1 −1.5 2.76507E−08 0.928 CDBox 128 HBII-202 −1.0 2.89826E−08 0.946 CDBox 135 miR-768-5p −1.6  3.1365E−08 0.986 miRNA 135 miR-421 1.6 4.42916E−08 0.872 miRNA 68 miR-28-5p 1.7 5.45027E−08 0.877 miRNA 105 miR-151-5p 1.2 5.48542E−08 0.968 miRNA 135

TABLE 5 miRNAs significantly expressed between metastatic melanoma and melanoma Log2- #Sample Probe Name FC P value AUC ProbeType Detected miR-31 −2.4 6.23649E−09 0.891 miRNA 103 miR-150 −1.2 4.84574E−06 0.783 miRNA 117 miR-203 −2.0 2.77555E−05 0.764 miRNA 132 ENSG00000212139_x 0.6 2.83257E−05 0.799 snoRNA 124 mgU6-53 0.6  3.8628E−05 0.828 CDBox 94 U72_x 0.5 4.47709E−05 0.783 HAcaBox 103 U94 0.6 7.34024E−05 0.776 CDBox 109 HBII-85-15_x 0.8 0.000176472 0.767 CDBox 49 HBII-85-29_x 0.8 0.000274959 0.734 CDBox 85 HBII-55 0.5 0.000331716 0.806 CDBox 135 snR38B 0.8 0.000374886 0.750 CDBox 126 miR-200c −1.6 0.00042063 0.718 miRNA 134 U61 0.7 0.000440758 0.785 CDBox 116 HBII-316 0.6 0.000548042 0.744 CDBox 130 U32A_x 0.4 0.000553879 0.748 CDBox 136 U81_x 0.6 0.000598062 0.720 CDBox 120 U15A 0.7 0.000649777 0.785 CDBox 128 ACA14b_x 0.4 0.000764147 0.748 HAcaBox 105 miR-182 −0.9 0.000868427 0.762 miRNA 113 miR-455-3p −0.7 0.000874654 0.718 miRNA 136 miR-532-5p −0.8 0.000883675 0.752 miRNA 113 mgU6-53_x 0.5 0.001038416 0.783 CDBox 113 ACA46 0.4 0.001076758 0.746 HAcaBox 124 miR-1234 −0.5 0.001112272 0.756 miRNA 68 U53 0.6 0.001130502 0.769 CDBox 126 HBII-85-29 0.7 0.001192467 0.702 CDBox 82 U47 0.5 0.001218674 0.702 CDBox 74 U42B_x 0.5 0.001412559 0.735 CDBox 109 miR-155 −1.3 0.001425842 0.716 miRNA 123 U64 0.5 0.001493551 0.728 HAcaBox 101 ACA28 0.5 0.001565276 0.692 HAcaBox 120 U13 0.6 0.001630341 0.748 CDBox 135 U84 0.4 0.001673776 0.769 CDBox 131 U22 0.5 0.001739769 0.714 CDBox 135 U46_x 0.6 0.001758635 0.755 CDBox 131 ACA17_x 0.6 0.001827243 0.705 HAcaBox 44 ENSG00000200897 0.4 0.001857043 0.735 snoRNA 38 U36A_x 0.4 0.00192703 0.728 CDBox 133 HBII-180C 0.4 0.001987135 0.757 CDBox 115 HBII-251 0.4 0.0020213 0.741 CDBox 135 ACA49 0.4 0.002175278 0.744 HAcaBox 127 U19 0.6 0.00221226 0.696 HAcaBox 118 miR-940 −0.6 0.002377881 0.713 miRNA 53 U76 0.6 0.002408733 0.755 CDBox 135 HBII-13_x 0.7 0.002521867 0.730 CDBox 60 HBII-382_s 0.6 0.002554678 0.770 scaRna 126 miR-205 −1.1 0.002610442 0.603 miRNA 135 U21 0.7 0.002613305 0.774 CDBox 134 miR-342-3p −0.5 0.002621512 0.706 miRNA 136 ENSG00000212326 0.5 0.002879558 0.728 snoRNA 104 U82 0.7 0.002979246 0.828 CDBox 131 HBII-234_x 0.3 0.003087142 0.734 CDBox 102 HBII-85-24_x 0.4 0.003172633 0.703 CDBox 34 U101 0.5 0.003644287 0.672 CDBox 134 U46 0.5 0.003690792 0.747 CDBox 133 U58A 0.5 0.003778891 0.735 CDBox 129 HBII-210 0.5 0.003785668 0.767 CDBox 135 U102 0.4 0.00401337 0.705 CDBox 124 HBII-85-3_x 0.5 0.004030857 0.694 CDBox 72 U92 0.4 0.004575813 0.712 scaRna 129 spike_in-control-31 0.1 0.004779896 0.705 Oligonucleotide 137 spike-in controls snR38C 0.6 0.005413855 0.779 CDBox 134 HBII-99 0.5 0.005634509 0.714 CDBox 122 ACA47 0.5 0.006095831 0.696 scaRna 77 HBII-420 0.6 0.006404916 0.824 CDBox 131 ENSG00000212423_x 0.5 0.006424898 0.684 snoRNA 115 HBII-85-17_x 0.6 0.006577979 0.678 CDBox 57 ACA27_x 0.3 0.006580858 0.725 HAcaBox 130 U79 0.6 0.007498133 0.748 CDBox 134 U107 0.4 0.00761608 0.707 HAcaBox 129 HBI-43 0.3 0.007872874 0.691 CDBox 122 U49B_x 0.5 0.008037057 0.736 CDBox 122 miR-548i −0.5 0.008242048 0.684 miRNA 36 miR-500-star −0.6 0.008434274 0.697 miRNA 97 HBII-85-2_x 0.4 0.008511275 0.708 CDBox 135 ACA41 0.5 0.008991193 0.723 HAcaBox 115 ACA3 0.5 0.009231708 0.737 HAcaBox 134 U49A 0.6 0.009381377 0.745 CDBox 135 HBII-85-20_x 0.5 0.00942941 0.672 CDBox 42 ENSG00000212508 −0.5 0.009471802 0.712 snoRNA 122 U38B_x 0.7 0.009547747 0.837 CDBox 133 ACA51_x 0.3 0.009599762 0.686 HAcaBox 135 ENSG00000200879 0.5 0.009860049 0.697 snoRNA 120 miR-181a −0.6 0.009890715 0.730 miRNA 134 ENSG00000200932 0.4 0.010075893 0.740 snoRNA 47 miR-589-star −0.4 0.010240715 0.650 miRNA 43 ENSG00000199262 0.4 0.010309025 0.694 snoRNA 59 miR-1324 −0.5 0.010648472 0.724 miRNA 57 U91_s 0.5 0.010903574 0.695 scaRna 132 U3-2_s 0.5 0.011269742 0.729 CDBox 135 U51_x 0.4 0.011519055 0.713 CDBox 130 ACA9 0.5 0.012046848 0.703 HAcaBox 82 miR-1825 −0.8 0.01292318 0.738 miRNA 132 mgU6-47 0.4 0.013536315 0.710 CDBox 47 miR-1281 −0.7 0.014086281 0.701 miRNA 137 U71d_x 0.5 0.01413005 0.678 HAcaBox 99 ACA42 0.4 0.014283243 0.786 HAcaBox 117 ACA34 0.3 0.014399902 0.637 HAcaBox 109 U48 0.4 0.014650847 0.654 CDBox 129 ENSG00000202093 0.5 0.014675533 0.689 snoRNA 97 miR-665 0.5 0.014853517 0.678 miRNA 47 miR-141 −0.7 0.015316578 0.647 miRNA 69 U23 0.4 0.015408258 0.701 HAcaBox 127 U15B 0.4 0.015684928 0.719 CDBox 129 miR-1274b 0.6 0.01695735 0.679 miRNA 112 U31_x 0.5 0.017818548 0.771 CDBox 135 ENSG00000200394_x 0.4 0.017859341 0.682 snoRNA 115 ACA67_x 0.4 0.018242565 0.663 HAcaBox 54 U80 0.4 0.019116778 0.683 CDBox 132 HBII-85-18_x 0.4 0.019329528 0.661 CDBox 48 U106 0.3 0.019924806 0.653 CDBox 112 U99 0.3 0.01993828 0.674 HAcaBox 137 U24 0.3 0.020062119 0.673 CDBox 133 miR-20a-star 1.0 0.020518991 0.694 miRNA 61 U16 0.4 0.0213066 0.669 CDBox 131 U49B_s 0.5 0.0213349 0.716 CDBox 129 miR-885-3p 0.7 0.021367498 0.694 miRNA 73 U36B 0.4 0.021413004 0.669 CDBox 121 HBII-295 0.3 0.021589703 0.678 CDBox 128 miR-148b 0.6 0.021711762 0.661 miRNA 32 U38B 0.7 0.022451358 0.834 CDBox 134 U31 0.6 0.023045818 0.738 CDBox 132 ACA8_x 0.4 0.023508719 0.697 HAcaBox 125 HBI-6_x 0.5 0.02438732 0.675 HAcaBox 115 mgU6-53B 0.3 0.024830223 0.687 CDBox 99 ACA62 0.4 0.024948935 0.714 HAcaBox 88 miR-23b −0.5 0.025010006 0.658 miRNA 137 mgh18S-121 0.4 0.02717981 0.773 CDBox 134 miR-134 −0.6 0.027498011 0.648 miRNA 61 ENSG00000202216 −0.3 0.02787169 0.654 snoRNA 53 snR38A 0.5 0.028058616 0.706 CDBox 130 ACA54 0.4 0.028079033 0.741 HAcaBox 133 U83 0.4 0.028849489 0.733 CDBox 135 U56_x 0.5 0.03056515 0.673 CDBox 134 ENSG00000207118 0.5 0.030731527 0.642 snoRNA 93 U71b_x 0.3 0.031331331 0.639 HAcaBox 117 U104 0.5 0.031533136 0.750 CDBox 135 miR-26b 0.6 0.031546885 0.666 miRNA 45 ENSG00000201129 0.4 0.031647545 0.658 snoRNA 54 HBII-180C_x 0.3 0.031765824 0.686 CDBox 131 miR-1257 −0.4 0.032490122 0.676 miRNA 31 miR-1285 0.6 0.032817723 0.656 miRNA 54 ACA6 0.4 0.033332825 0.706 HAcaBox 126 HBII-166 0.3 0.033479679 0.715 CDBox 134 U20 0.3 0.033581135 0.659 CDBox 117 U49A_x 0.5 0.034235707 0.734 CDBox 135 HBII-85-11 0.5 0.034401945 0.626 CDBox 31 miR-198 0.5 0.035071443 0.673 miRNA 31 miR-1273 0.4 0.035126423 0.649 miRNA 47 U8_x 0.4 0.035369399 0.661 CDBox 135 miR-127-3p −0.5 0.035478749 0.646 miRNA 89 U36C 0.3 0.036400622 0.690 CDBox 135 miR-502-3p −0.5 0.036440109 0.696 miRNA 110 U97 0.4 0.036556318 0.713 CDBox 133 HBII-13 0.5 0.038037592 0.639 CDBox 52 miR-148a 0.6 0.038972124 0.647 miRNA 41 hsa-let-7b −0.7 0.040092161 0.713 miRNA 136 U55 0.3 0.041929175 0.687 CDBox 135 HBII-85-5_x 0.3 0.04234391 0.645 CDBox 31 U105 0.3 0.043199784 0.658 CDBox 126 U90 0.3 0.043617609 0.701 scaRna 114 ENSG00000202252 0.3 0.044418964 0.647 snoRNA 135 ENSG00000201467_x −0.3 0.04502792 0.628 snoRNA 31 ACA21 0.5 0.045481211 0.754 HAcaBox 133 U52 0.4 0.045645643 0.708 CDBox 135 U18C_x 0.4 0.046127928 0.637 CDBox 74 HBII-296B 0.3 0.046365102 0.657 CDBox 98 ENSG00000200307 0.3 0.048211492 0.613 snoRNA 86 miR-509-3p −1.2 0.048257749 0.574 miRNA 116 miR-1202 0.5 0.048318042 0.661 miRNA 57 ACA36_x 0.4 0.048990385 0.670 HAcaBox 88 ENSG00000207016_x 0.3 0.049618219 0.660 snoRNA 48 miR-30c 0.3 0.049698 0.709 miRNA 125 ACA19 0.3 0.049825075 0.655 HAcaBox 118

TABLE 6 50 miRNAs most significantly expressed between metastatic melanoma and melanoma Log2- Probe Name FC P value AUC ProbeType #Sample Detected miR-31 −2.4 6.23649E−09 0.891 miRNA 103 miR-150 −1.2 4.84574E−06 0.783 miRNA 117 miR-203 −2.0 2.77555E−05 0.764 miRNA 132 ENSG00000212139_x 0.6 2.83257E−05 0.799 snoRNA 124 mgU6-53 0.6  3.8628E−05 0.828 CDBox 94 U72_x 0.5 4.47709E−05 0.783 HAcaBox 103 U94 0.6 7.34024E−05 0.776 CDBox 109 HBII-85-15_x 0.8 0.000176472 0.767 CDBox 49 HBII-85-29_x 0.8 0.000274959 0.734 CDBox 85 HBII-55 0.5 0.000331716 0.806 CDBox 135 snR38B 0.8 0.000374886 0.750 CDBox 126 miR-200c −1.6 0.00042063 0.718 miRNA 134 U61 0.7 0.000440758 0.785 CDBox 116 HBII-316 0.6 0.000548042 0.744 CDBox 130 U32A_x 0.4 0.000553879 0.748 CDBox 136 U81_x 0.6 0.000598062 0.720 CDBox 120 U15A 0.7 0.000649777 0.785 CDBox 128 ACA14b_x 0.4 0.000764147 0.748 HAcaBox 105 miR-182 −0.9 0.000868427 0.762 miRNA 113 miR-455-3p −0.7 0.000874654 0.718 miRNA 136 miR-532-5p −0.8 0.000883675 0.752 miRNA 113 mgU6-53_x 0.5 0.001038416 0.783 CDBox 113 ACA46 0.4 0.001076758 0.746 HAcaBox 124 miR-1234 −0.5 0.001112272 0.756 miRNA 68 U53 0.6 0.001130502 0.769 CDBox 126 HBII-85-29 0.7 0.001192467 0.702 CDBox 82 U47 0.5 0.001218674 0.702 CDBox 74 U42B_x 0.5 0.001412559 0.735 CDBox 109 miR-155 −1.3 0.001425842 0.716 miRNA 123 U64 0.5 0.001493551 0.728 HAcaBox 101 ACA28 0.5 0.001565276 0.692 HAcaBox 120 U13 0.6 0.001630341 0.748 CDBox 135 U84 0.4 0.001673776 0.769 CDBox 131 U22 0.5 0.001739769 0.714 CDBox 135 U46_x 0.6 0.001758635 0.755 CDBox 131 ACA17_x 0.6 0.001827243 0.705 HAcaBox 44 ENSG00000200897 0.4 0.001857043 0.735 snoRNA 38 U36A_x 0.4 0.00192703 0.728 CDBox 133 HBII-180C 0.4 0.001987135 0.757 CDBox 115 HBII-251 0.4 0.0020213 0.741 CDBox 135 ACA49 0.4 0.002175278 0.744 HAcaBox 127 U19 0.6 0.00221226 0.696 HAcaBox 118 miR-940 −0.6 0.002377881 0.713 miRNA 53 U76 0.6 0.002408733 0.755 CDBox 135 HBII-13_x 0.7 0.002521867 0.730 CDBox 60 HBII-382_s 0.6 0.002554678 0.770 scaRna 126 miR-205 −1.1 0.002610442 0.603 miRNA 135 U21 0.7 0.002613305 0.774 CDBox 134 miR-342-3p −0.5 0.002621512 0.706 miRNA 136 ENSG00000212326 0.5 0.002879558 0.728 snoRNA 104

TABLE 7 miRNAs significantly expressed between nevi and normal skin Log2- #Sample Probe Name FC P value AUC ProbeType Detected 14qll-14 −3.87  3.6592E−27 1.000 CDBox 56 14qll-14_x −3.21  3.815E−24 1.000 CDBox 56 U74_x 1.46 3.89068E−23 1.000 CDBox 135 miR-509-3p 5.78 1.67661E−22 0.997 miRNA 116 miR-768-5p −1.46 5.23441E−22 1.000 miRNA 135 ENSG00000199411_s 1.36 1.56796E−21 1.000 snoRNA 135 Z17B −1.94 2.89347E−20 0.997 CDBox 117 miR-149 −3.46 1.55425E−19 1.000 miRNA 100 miR-125a-5p −3.44 1.86526E−19 1.000 miRNA 128 U43 −1.19 3.32514E−19 1.000 CDBox 135 miR-1308 1.55  8.1184E−19 0.964 miRNA 135 U59A 1.13 9.99768E−18 0.993 CDBox 135 miR-146a 4.44 3.16232E−17 0.975 miRNA 129 miR-513a-5p 3.77 1.05679E−16 0.972 miRNA 105 ACA20 −1.74 1.56297E−16 0.993 HAcaBox 131 U43_x −1.05 1.04459E−15 1.000 CDBox 135 U44_x −1.37 5.54123E−15 0.994 CDBox 135 miR-127-3p −3.56 7.53549E−15 0.988 miRNA 89 miR-193b −2.57 7.81928E−15 1.000 miRNA 130 U83 −1.25 9.30493E−15 0.993 CDBox 135 miR-768-3p 0.98 2.39925E−14 0.972 miRNA 137 U33 −1.02 2.44194E−14 0.997 CDBox 137 miR-574-3p −3.05  3.0433E−14 0.974 miRNA 122 ACA16 −1.86 3.35128E−14 0.968 HAcaBox 47 14ql-4 −2.43 3.96317E−14 0.979 CDBox 80 hsa-let-7c −1.01 4.99111E−14 0.986 miRNA 135 miR-342-3p −2.06 1.67074E−13 0.996 miRNA 136 hsa-let-7b −1.01 2.25904E−13 0.964 miRNA 136 miR-423-3p −2.52 2.97967E−13 1.000 miRNA 107 HBII-239 −1.41  3.2162E−13 0.975 CDBox 135 U38B_x 1.52 3.80613E−13 0.968 CDBox 133 miR-320b −0.63 6.31909E−13 0.971 miRNA 135 miR-923 0.93 6.48332E−13 0.990 miRNA 135 U54 −1.06 1.17197E−12 0.975 CDBox 135 U38B 1.80 1.18885E−12 0.976 CDBox 134 U44 −1.07 1.28887E−12 0.981 CDBox 134 miR-139-5p −2.70 2.74261E−12 0.940 miRNA 80 miR-1826 −0.71 2.83317E−12 0.958 miRNA 137 ENSG00000199435 1.38 2.88249E−12 0.954 snoRNA 63 miR-26a 0.89 2.88328E−12 0.960 miRNA 136 U58B_x −1.47 3.60008E−12 0.940 CDBox 132 HBII-180A_x −1.43 4.25614E−12 0.992 CDBox 123 14qll-12_x −2.09 4.30741E−12 0.949 CDBox 85 miR-509-3-5p 3.46 5.53398E−12 0.953 miRNA 107 U55_x −1.35  6.7836E−12 0.996 CDBox 135 miR-149-star 0.93  9.6022E−12 0.940 miRNA 135 HBII-276 −1.41 1.19464E−11 0.964 CDBox 132 miR-23b −0.75 1.72105E−11 0.968 miRNA 137 miR-15b −2.42  1.7823E−11 0.968 miRNA 125 miR-921 1.73 1.93285E−11 0.954 miRNA 49 miR-191 −0.85  2.0881E−11 0.972 miRNA 135 U103_s −1.60 2.57483E−11 0.943 CDBox 111 miR-486-5p −3.27 2.90873E−11 0.942 miRNA 65 U27 −1.61 3.28995E−11 0.960 CDBox 134 miR-1202 2.18 4.44562E−11 0.945 miRNA 57 U49A 1.25 4.51441E−11 0.970 CDBox 135 ACA24_x −1.50 5.61856E−11 0.922 HAcaBox 98 HBII-382_s 1.30 5.89071E−11 0.953 scaRna 126 U52 −0.92 1.05668E−10 0.976 CDBox 135 miR-99b −1.84 1.06853E−10 0.988 miRNA 129 U38A 0.84 1.30902E−10 0.936 CDBox 134 U46 −1.44 1.33613E−10 0.965 CDBox 133 U57 −1.10 1.66897E−10 0.967 CDBox 135 U3-2_s 0.70 1.79537E−10 0.925 CDBox 135 14qll-26_x −1.60 1.92804E−10 0.922 CDBox 41 ENSG00000207098_x 1.21 2.17044E−10 0.956 snoRNA 86 miR-320c −0.84 2.33286E−10 0.994 miRNA 135 miR-199a-5p −2.53 2.85196E−10 0.994 miRNA 129 HBII-180C −1.55 3.80017E−10 0.924 CDBox 115 U63 0.94 4.46838E−10 0.945 CDBox 135 HBII-436 −1.22 4.98357E−10 0.929 CDBox 102 U81_x −1.25 6.27975E−10 0.928 CDBox 120 HBII-419 1.10 6.92884E−10 0.932 CDBox 133 U17b −1.32 8.76094E−10 0.996 HAcaBox 135 miR-506 2.11 1.46252E−09 0.931 miRNA 76 miR-1268 1.02 1.54826E−09 0.909 miRNA 133 miR-671-5p 1.38 1.60795E−09 0.914 miRNA 69 miR-339-5p −2.03 1.92249E−09 0.907 miRNA 75 ACA48_x −1.25 1.98889E−09 0.945 HAcaBox 130 14qll-21_x −1.20 2.04198E−09 0.907 CDBox 51 ENSG00000202093_x −1.37 2.13676E−09 0.902 snoRNA 125 U73a −0.76 3.11274E−09 0.924 CDBox 135 miR-1228-star 1.09 3.32612E−09 0.925 miRNA 134 miR-193b-star −1.99 3.54373E−09 0.925 miRNA 77 miR-145 −2.87 3.67076E−09 0.986 miRNA 135 miR-1207-5p 0.91 3.82606E−09 0.936 miRNA 133 miR-23a −0.62 4.38608E−09 0.950 miRNA 137 14qll-3 −1.37 5.04275E−09 0.938 CDBox 95 U56 0.82 5.34057E−09 0.910 CDBox 135 miR-320a −0.50 5.87182E−09 0.917 miRNA 135 HBII-289 −0.82 6.53343E−09 0.975 CDBox 135 mgU6-53B 1.17 6.66322E−09 0.946 CDBox 99 miR-222 −1.42 6.66965E−09 1.000 miRNA 135 miR-135a-star 1.78 6.83122E−09 0.914 miRNA 44 HBII-142 0.50 6.91032E−09 0.911 CDBox 135 14qll-12 −1.90  7.3584E−09 0.896 CDBox 58 ACA23 −1.07 9.00722E−09 0.922 HAcaBox 113 miR-1224-5p 2.00 9.14764E−09 0.902 miRNA 78 U17b_x −0.78 9.16941E−09 0.917 HAcaBox 135 U41 −0.89  1.0052E−08 0.928 CDBox 135 U55 −1.40 1.07645E−08 0.988 CDBox 135 U101 1.01 1.22325E−08 0.911 CDBox 134 miR-193a-5p −2.09 1.43928E−08 0.954 miRNA 121 miR-720 0.87 1.45059E−08 0.918 miRNA 137 miR-205 −1.04 1.53388E−08 0.956 miRNA 135 mgh28S-2411 −0.86 1.81685E−08 0.965 CDBox 135 miR-1300 1.84 1.83075E−08 0.911 miRNA 32 miR-22 −2.31 1.87992E−08 0.943 miRNA 120 ACA40_x −1.14 1.89306E−08 0.935 HAcaBox 134 miR-92a −1.48 1.96388E−08 1.000 miRNA 136 14qll-28_x −1.20 2.08991E−08 0.911 CDBox 51 U23 −0.93 2.35134E−08 0.913 HAcaBox 127 snR38C 0.62 2.69211E−08 0.902 CDBox 134 miR-939 1.33  2.8172E−08 0.947 miRNA 57 miR-214 −0.88 2.85157E−08 0.921 miRNA 135 U94 1.03 3.14773E−08 0.898 CDBox 109 miR-510 2.20 3.61106E−08 0.899 miRNA 82 miR-181b −1.85 5.28195E−08 0.961 miRNA 130 U15B −1.06 5.57521E−08 0.898 CDBox 129 gi555853_copy5 −0.51 7.13438E−08 0.910 5.8s rRNA 137 miR-432 −1.83 7.49361E−08 0.902 miRNA 51 HBII-142_x 0.47 8.53374E−08 0.886 CDBox 135 snR38B 1.11 8.92959E−08 0.899 CDBox 126 ACA36_x 1.12 9.60154E−08 0.885 HAcaBox 88 miR-320d −1.66 1.03365E−07 0.997 miRNA 134 hsa-let-7i 1.72 1.07923E−07 0.947 miRNA 135 miR-125b-2-star −1.66 1.09644E−07 0.881 miRNA 58 miR-572 1.35 1.13617E−07 0.906 miRNA 116 U102 0.87  1.2385E−07 0.874 CDBox 124 gi555853_copy8 −0.51 1.25699E−07 0.903 5.8s rRNA 137 miR-371-5p 1.93 1.26302E−07 0.886 miRNA 64 miR-125b −0.97 1.35947E−07 0.988 miRNA 135 ENSG00000201619 1.40  1.4629E−07 0.892 snoRNA 62 U78_x 1.11 1.62568E−07 0.921 CDBox 134 ENSG00000212397 0.90 1.65415E−07 0.898 snoRNA 134 ENSG00000212182 1.09 1.80486E−07 0.884 snoRNA 59 hsa-let-7f 1.99  2.0615E−07 0.909 miRNA 130 HBII-55 −0.82 2.17315E−07 0.940 CDBox 135 gi555853_copy2 −0.49 2.37022E−07 0.893 5.8s rRNA 137 ACA15_s −1.09 2.48572E−07 0.875 HAcaBox 116 U28_x −0.90  2.5645E−07 0.896 CDBox 133 gi555853_copy0 −0.50 2.65495E−07 0.889 5.8s rRNA 137 gi555853_copy4 −0.48 2.85254E−07 0.898 5.8s rRNA 137 ENSG00000201816 0.83 3.19058E−07 0.878 snoRNA 49 gi555853_copy1 −0.48 3.26181E−07 0.886 5.8s rRNA 137 U60 −0.97 3.26261E−07 0.871 CDBox 103 gi555853_copy6 −0.47 3.40126E−07 0.900 5.8s rRNA 137 miR-132 −1.77 3.42625E−07 0.870 miRNA 96 U68_x −1.05 3.51611E−07 0.910 HAcaBox 133 gi555853_copy7 −0.48 4.04469E−07 0.892 5.8s rRNA 137 ENSG00000201660 1.12 4.27296E−07 0.896 snoRNA 132 miR-150-star 1.63 4.51511E−07 0.874 miRNA 48 miR-665 1.55 5.14634E−07 0.871 miRNA 47 miR-150 −1.60  6.5278E−07 0.884 miRNA 117 miR-210 −1.24 6.58793E−07 0.911 miRNA 130 miR-664-star 1.38 6.79224E−07 0.864 miRNA 96 U49A_x 1.03 7.09035E−07 0.911 CDBox 135 miR-30c −1.75 7.10279E−07 0.911 miRNA 125 ENSG00000212458 0.92 7.71477E−07 0.871 snoRNA 95 ENSG00000200879 −1.08 7.74449E−07 0.868 snoRNA 120 hsa-let-7d 0.39 8.11829E−07 0.886 miRNA 135 14qll-1_x −1.24 9.36723E−07 0.899 CDBox 131 14qll-26 −1.14 1.46915E−06 0.827 CDBox 32 hsa-let-7a 0.51 1.65358E−06 0.841 miRNA 136 U28 −0.75 1.71566E−06 0.874 CDBox 135 U68 −0.88 1.83186E−06 0.892 HAcaBox 130 HBII-180C_x −0.95 2.06263E−06 0.882 CDBox 131 gi555853_copy3 −0.45 2.14632E−06 0.863 5.8s rRNA 137 miR-2001b-star −1.70 2.20935E−06 0.861 miRNA 74 miR-1234 1.03  2.2168E−06 0.861 miRNA 68 U95 −0.36 2.37891E−06 0.852 CDBox 135 U46_x −0.98 2.42909E−06 0.873 CDBox 131 spike_in-control-21 0.74 2.57369E−06 0.827 Oligonucleotide 42 spike-in controls miR-1248 1.03 3.51695E−06 0.874 miRNA 39 U36A −0.61 3.61256E−06 0.882 CDBox 132 U13 0.50 4.36581E−06 0.834 CDBox 135 miR-92a-2-star 1.02 4.99387E−06 0.859 miRNA 46 miR-663 0.77 5.24563E−06 0.859 miRNA 134 miR-409-3p −1.42 5.63777E−06 0.837 miRNA 92 U34 −0.68 5.88695E−06 0.852 CDBox 137 U25 −0.71 6.25404E−06 0.896 CDBox 135 U32A_x −0.75 6.51615E−06 0.882 CDBox 136 U96a_x 0.68 6.70342E−06 0.849 CDBox 134 gi555853_copy9 −0.42  7.0704E−06 0.863 5.8s rRNA 137 ENSG00000199411_x 0.77 7.82781E−06 0.846 snoRNA 132 spike_in-control-7 1.00 7.92241E−06 0.848 Oligonucleotide 54 spike-in controls miR-198 1.24 8.11341E−06 0.846 miRNA 31 14ql-9_x −0.81 8.29164E−06 0.820 CDBox 34 miR-425 −1.72 8.48515E−06 0.846 miRNA 114 HBII-82 0.77 8.59528E−06 0.839 CDBox 90 miR-1180 −1.22 9.75327E−06 0.828 miRNA 37 ENSG00000206637_x 0.74 1.15035E−05 0.850 snoRNA 59 miR-502-3p −1.41 1.18784E−05 0.831 miRNA 110 4qll-22_x −0.97 1.49319E−05 0.792 CDBox 50 ACA18_x −0.72 1.77602E−05 0.837 HAcaBox 135 miR-1274b 1.66 1.82625E−05 0.813 miRNA 112 HBII-99 −0.94 1.84996E−05 0.837 CDBox 122 U35B 0.78 1.93006E−05 0.837 CDBox 128 ACA20_x −0.84 2.02452E−05 0.882 HAcaBox 134 miR-143 −2.21  2.0661E−05 0.864 miRNA 126 ENSG00000200652 0.81 2.09666E−05 0.824 snoRNA 40 HBII-202 −0.59 2.32067E−05 0.886 CDBox 135 miR-708 −1.81 2.34758E−05 0.816 miRNA 102 miR-182 −1.64 2.56273E−05 0.791 miRNA 113 miR-345 −1.28  3.1705E−05 0.816 miRNA 75 ACA62 0.82 3.35475E−05 0.817 HAcaBox 88 miR-1273 1.12 3.38999E−05 0.827 miRNA 47 ACA24_s −1.10 3.44078E−05 0.823 HAcaBox 128 miR-1272 0.97 3.51756E−05 0.820 miRNA 67 14ql-4_x −1.11 3.64993E−05 0.796 CDBox 51 14qll-9_x −0.89 3.72476E−05 0.820 CDBox 36 U106 −0.62 3.79813E−05 0.817 CDBox 112 miR-1285 1.22 4.16925E−05 0.796 miRNA 54 ENSG00000200492 0.77 4.61258E−05 0.838 snoRNA 60 ENSG00000202327 0.77 4.61736E−05 0.812 snoRNA 54 miR-126 1.24 4.86215E−05 0.845 miRNA 133 U49B_x 0.62 4.92521E−05 0.795 CDBox 122 ENSG00000207016_x 0.74  5.4547E−05 0.794 snoRNA 48 HBII-52-25_x 0.68 5.50793E−05 0.827 CDBox 57 U49B_s 0.90 5.94839E−05 0.848 CDBox 129 U53 −0.97 6.07982E−05 0.809 CDBox 126 miR-1246 1.19 7.10577E−05 0.799 miRNA 131 ENSG00000212615_x −0.67 7.76933E−05 0.798 snoRNA 102 miR-575 1.09 8.31223E−05 0.794 miRNA 41 snR38A 0.69 8.40239E−05 0.807 CDBox 130 14ql-7 −0.78 8.70579E−05 0.825 CDBox 51 miR-29b-2-star 0.86  8.9139E−05 0.802 miRNA 126 U80 −0.60 9.75089E−05 0.825 CDBox 132 miR-1225-5p 1.04 9.99249E−05 0.830 miRNA 90 miR-92b-star 0.94 0.000102055 0.809 miRNA 111 ACA53 −0.61 0.000103826 0.814 HAcaBox 105 ACA55 0.79 0.000105025 0.805 HAcaBox 85 U51 0.58 0.000112048 0.802 CDBox 134 U108_x 0.55 0.000112181 0.798 HAcaBox 87 U48 −1.07 0.000118819 0.875 CDBox 129 miR-221 −1.06 0.00013613 0.911 miRNA 135 HBII-13 0.74 0.000139215 0.798 CDBox 52 miR-19b 1.53 0.0001409 0.795 miRNA 124 ACA3-2 −0.76 0.000143105 0.819 HAcaBox 135 HBII-85-23_x −0.73 0.000143482 0.787 CDBox 58 14ql-8 −0.75 0.000167587 0.802 CDBox 61 miR-138-1-star 0.88 0.000190948 0.798 miRNA 91 spike_in-control-28 0.63 0.000200982 0.802 Oligonucleotide 76 spike-in controls miR-500-star −1.36 0.000204792 0.803 miRNA 97 ENSG00000212423_x 0.75 0.000232739 0.789 snoRNA 115 ENSG00000201348 0.50 0.000243216 0.778 snoRNA 94 miR-1275 1.09 0.000244874 0.821 miRNA 127 miR-30a-star −1.14 0.000247652 0.758 miRNA 64 miR-93-star −1.16 0.000248272 0.777 miRNA 48 ACA25 −0.64 0.000248534 0.802 HAcaBox 73 U50 −0.73 0.000255129 0.853 CDBox 134 14qll-1 −1.00 0.000255606 0.807 CDBox 128 U14B_x −0.79 0.000258746 0.788 CDBox 53 miR-638 0.35 0.000261809 0.823 miRNA 135 U51_x 0.57 0.000299812 0.820 CDBox 130 miR-99a −0.87 0.000321188 0.832 miRNA 135 miR-491-5p 1.04 0.000341416 0.756 miRNA 92 miR-508-3p 1.23 0.000350578 0.778 miRNA 74 miR-508-5p 1.02 0.000377448 0.783 miRNA 92 miR-641 0.88 0.000391728 0.774 miRNA 39 miR-885-5p −1.23 0.00039874 0.801 miRNA 100 miR-497 −1.48 0.00041742 0.780 miRNA 104 U31_x −0.65 0.000420569 0.795 CDBox 135 miR-498 0.85 0.000424439 0.787 miRNA 56 miR-20a 1.21 0.000429079 0.807 miRNA 134 miR-509-5p 1.35 0.000476275 0.776 miRNA 81 miR-331-3p −1.11 0.000481237 0.777 miRNA 64 HBII-295 0.53 0.000526137 0.785 CDBox 128 ACA27_x −0.45 0.000528795 0.778 HAcaBox 130 miR-155 −1.29 0.000530869 0.751 miRNA 123 miR-652 −1.30 0.000593692 0.774 miRNA 119 ENSG00000208308_x −0.58 0.000595144 0.758 snoRNA 129 miR-1281 0.98 0.000606821 0.766 miRNA 137 E3_x −0.53 0.000619964 0.819 HAcaBox 135 miR-27a-star −0.88 0.000656179 0.773 miRNA 40 miR-92b −0.90 0.0006705 0.773 miRNA 91 miR-570 0.93 0.000670846 0.776 miRNA 88 miR-181a-2-star −1.19 0.000676824 0.753 miRNA 99 miR-1185 0.76 0.000680122 0.752 miRNA 42 ACA33 0.57 0.000718022 0.780 HAcaBox 133 ENSG00000212401 0.57 0.000773614 0.799 snoRNA 41 U20 −0.51 0.000816772 0.758 CDBox 117 U19 0.84 0.00082895 0.759 HAcaBox 118 miR-203 0.69 0.000839225 0.878 miRNA 132 ENSG00000212508 0.83 0.000873066 0.801 snoRNA 122 ACA54 −0.54 0.000880465 0.817 HAcaBox 133 ACA9_x −0.68 0.001025389 0.752 HAcaBox 77 14ql-1 0.61 0.001136212 0.729 CDBox 70 miR-940 0.87 0.001164857 0.737 miRNA 53 U14B −0.69 0.001178164 0.738 CDBox 51 mgU6-77 −0.62 0.001238112 0.760 CDBox 129 spike_in-control-30 0.54 0.001290622 0.753 Oligonucleotide 53 spike-in controls miR-629-star −0.92 0.00138462 0.756 miRNA 46 ACA49 0.56 0.001394857 0.791 HAcaBox 127 miR-483-5p 0.96 0.001461794 0.738 miRNA 48 ENSG00000212214_x 0.51 0.001505107 0.748 snoRNA 88 miR-574-5p −0.99 0.00156768 0.766 miRNA 103 miR-28-3p −1.29 0.001658961 0.747 miRNA 94 U36C 0.34 0.001694833 0.849 CDBox 135 miR-1288 0.73 0.001697813 0.738 miRNA 43 ACA37_x −0.59 0.001721183 0.742 HAcaBox 84 miR-1280 −0.72 0.001869035 0.777 miRNA 134 miR-487b −0.96 0.001988497 0.731 miRNA 58 miR-532-3p −0.80 0.00199381 0.766 miRNA 95 miR-346 −0.91 0.0020172 0.749 miRNA 55 miR-27b-star −1.04 0.00209985 0.742 miRNA 51 ACA32 0.34 0.002161552 0.785 HAcaBox 135 ENSG00000212206_x 0.58 0.002337296 0.740 snoRNA 74 ACA51_x 0.43 0.002449336 0.742 HAcaBox 135 ENSG00000201847_x −0.53 0.002461896 0.729 snoRNA 48 miR-513c 1.13 0.002510473 0.749 miRNA 73 miR-455-3p −0.62 0.00258213 0.785 miRNA 136 miR-188-5p −0.90 0.002825937 0.730 miRNA 51 ENSG00000207027 0.48 0.002903474 0.704 snoRNA 36 ENSG00000202498_x 0.56 0.002908782 0.735 snoRNA 137 HBII-85-26 0.66 0.003001767 0.741 CDBox 136 miR-337-3p 0.80 0.003015225 0.740 miRNA 94 U35A −0.47 0.003204612 0.723 CDBox 135 miR-106b-star −1.10 0.003210131 0.717 miRNA 72 ENSG00000202216 0.55 0.003292357 0.758 snoRNA 53 HBII-316 0.63 0.003333247 0.705 CDBox 130 miR-31 −1.63 0.003410838 0.720 miRNA 103 U22 0.43 0.003510102 0.699 CDBox 135 miR-34a-star 0.77 0.003765001 0.715 miRNA 39 HBII-85-6_x 0.62 0.003870576 0.735 CDBox 137 ACA15_x −0.50 0.003952375 0.734 HAcaBox 124 ENSG00000200706_x 0.48 0.00407636 0.762 snoRNA 43 ENSG00000212627 0.41 0.00409948 0.726 snoRNA 69 14qll-7 0.59 0.004220389 0.719 CDBox 77 ENSG00000212432_s 0.52 0.004352612 0.745 snoRNA 75 miR-214-star −0.82 0.004514439 0.708 miRNA 47 miR-106a 0.58 0.004682448 0.724 miRNA 135 ACA42 −0.54 0.004759271 0.720 HAcaBox 117 mgU6-53B_x 0.46 0.0048197 0.720 CDBox 121 ACA19 0.38 0.005153495 0.733 HAcaBox 118 ACA50 −0.53 0.005300761 0.738 HAcaBox 88 miR-138 1.07 0.005618175 0.716 miRNA 82 U47 −0.47 0.005755719 0.729 CDBox 74 ENSG00000212551 0.48 0.005885716 0.740 snoRNA 73 miR-769-5p −0.93 0.005951319 0.687 miRNA 56 miR-1228 0.82 0.006114287 0.724 miRNA 114 miR-30b 0.87 0.00634299 0.748 miRNA 128 ENSG00000212315 0.54 0.006470787 0.752 snoRNA 117 ENSG00000212538 0.48 0.006558923 0.735 snoRNA 35 ACA2b 0.39 0.007073038 0.756 HAcaBox 47 ENSG00000212523_x 0.59 0.007115341 0.716 snoRNA 135 miR-196a 1.04 0.007138032 0.705 miRNA 77 HBII-429 0.22 0.007262326 0.720 CDBox 137 miR-16 0.46 0.007360456 0.749 miRNA 135 mgh28S-2409 0.27 0.007642791 0.741 CDBox 135 ENSG00000200307_x 0.54 0.007656242 0.695 snoRNA 50 U76 −0.27 0.008469646 0.734 CDBox 135 14qll-17 −0.54 0.008809376 0.698 CDBox 71 miR-324-3p −0.67 0.009111464 0.726 miRNA 100 ACA48 −0.49 0.009193835 0.798 HAcaBox 121 miR-200c −0.31 0.009670586 0.625 miRNA 134 miR-1260 −0.78 0.010502121 0.726 miRNA 120 miR-130a −0.94 0.010557093 0.687 miRNA 122 U17a −0.52 0.010593144 0.702 HAcaBox 105 ACA52 −0.45 0.010843862 0.708 HAcaBox 121 miR-197 −0.79 0.011117393 0.724 miRNA 103 U66 −0.54 0.011246742 0.705 HAcaBox 119 ENSG00000202252 −0.30 0.011273976 0.699 snoRNA 135 ACA61 0.22 0.011490951 0.730 HAcaBox 135 miR-151-5p 0.56 0.01153084 0.830 miRNA 135 ACA16_x −0.47 0.011881173 0.727 HAcaBox 98 U18C_x −0.45 0.012073238 0.691 CDBox 74 U15A 0.45 0.012634875 0.706 CDBox 128 miR-1274a 0.71 0.012698242 0.684 miRNA 48 ACA −0.62 0.01271037 0.691 HAcaBox 82 U91_s −0.54 0.012949986 0.733 scaRna 132 ENSG00000207002 −0.52 0.013212929 0.702 snoRNA 35 miR-129-3p −0.54 0.013303701 0.705 miRNA 59 spike_in-cortrol-17 0.48 0.013556785 0.705 Oligonucleotide 33 spike-in controls spike_in-control-2 0.19 0.014699249 0.684 Oligonucleotide 137 spike-in controls ENSG00000212206 0.49 0.014866744 0.670 snoRNA 46 miR-107 −0.39 0.015131674 0.669 miRNA 135 ACA63 0.42 0.01519582 0.695 HAcaBox 66 ENSG00000212553_x 0.45 0.015776333 0.684 snoRNA 60 U50B −0.37 0.016500241 0.704 CDBox 135 ENSG00000207100_x 0.36 0.016825837 0.666 snoRNA 72 U78_s −0.55 0.016840855 0.702 CDBox 135 miR-551b-star −0.65 0.016907402 0.686 miRNA 61 ACA47 0.45 0.017340107 0.666 scaRna 77 U92 0.45 0.017435918 0.760 scaRna 129 miR-342-5p −0.78 0.01779742 0.695 miRNA 98 miR-128 −0.56 0.017820912 0.699 miRNA 44 U17a_x −0.46 0.018370462 0.720 HAcaBox 115 ENSG00000206909_x 0.44 0.018373221 0.706 snoRNA 47 U30 −0.35 0.018607695 0.729 CDBox 135 miR-98 −0.57 0.019510485 0.677 miRNA 47 miR-21-star 0.62 0.019769732 0.686 miRNA 64 miR-135b-star 0.57 0.0198019 0.704 miRNA 38 ACA2a −0.42 0.019986353 0.677 HAcaBox 55 U88 0.44 0.020351208 0.672 scaRna 69 HBII-85-29_x 0.39 0.020664805 0.666 CDBox 85 14qll-19 0.47 0.02089152 0.695 CDBox 43 U83B 0.19 0.020956657 0.677 CDBox 135 miR-134 −0.86 0.020986754 0.681 miRNA 61 U83A −0.40 0.021328386 0.698 CDBox 127 ENSG00000202370 0.53 0.02172281 0.651 snoRNA 88 U75_x −0.35 0.021862862 0.697 HAcaBox 103 ACA38 −0.38 0.023364662 0.688 HAcaBox 42 ENSG00000200394_x 0.42 0.023498919 0.661 snoRNA 115 miR-1324 0.50 0.023518344 0.684 miRNA 57 miR-106b 0.85 0.023747964 0.670 miRNA 127 miR-589-star 0.56 0.023792146 0.675 miRNA 43 ACA6 −0.36 0.024014637 0.673 HAcaBox 126 ACA28 0.36 0.02403059 0.687 HAcaBox 120 ACA57 0.36 0.024679323 0.748 scaRna 135 HBII-85-4_x 0.36 0.024717662 0.711 CDBox 125 ENSG00000200897 −0.41 0.024775335 0.662 snoRNA 38 U16 0.42 0.024969966 0.712 CDBox 131 ENSG00000200418 −0.41 0.025790882 0.668 snoRNA 31 U105 −0.31 0.026442326 0.673 CDBox 126 spike_in-control-34 0.44 0.026481739 0.665 Oligonucleotide 58 spike-in controls spike_in-control-23 0.24 0.027477358 0.645 Oligonucleotide 137 spike-in controls miR-196a-star 0.61 0.028243677 0.677 miRNA 37 HBII-296A 0.33 0.02845867 0.659 CDBox 78 U82 0.43 0.028925335 0.681 CDBox 131 ENSG00000212558_x −0.35 0.029963606 0.662 snoRNA 31 miR-21 0.67 0.030060673 0.668 miRNA 79 ACA31 0.37 0.030929941 0.702 HAcaBox 123 miR-1257 0.55 0.031016296 0.676 miRNA 31 ACA14b_x −0.37 0.031018594 0.688 HAcaBox 105 ACA33_x 0.38 0.033957171 0.687 HAcaBox 129 miR-199b-3p 0.64 0.034530062 0.737 miRNA 134 miR-100 −0.61 0.034553062 0.681 miRNA 133 U64 −0.38 0.034971581 0.633 HAcaBox 101 miR-10a −0.67 0.037624151 0.666 miRNA 69 miR-30e 0.56 0.037770496 0.683 miRNA 57 miR-199a-3p 0.62 0.038490813 0.704 miRNA 134 HBII-85-11 −0.37 0.038507691 0.687 CDBox 31 miR-505-star −0.62 0.039242017 0.647 miRNA 58 hsa-let-7f-1-star 0.44 0.042251211 0.658 miRNA 64 spike_in-control-29 −0.19 0.043064083 0.684 Oligonucleotide 137 spike-in controls miR-20b 0.64 0.043397678 0.655 miRNA 99 ACA5 −0.40 0.044405124 0.693 HAcaBox 85 miR-628-3p 0.57 0.044447799 0.652 miRNA 38 HBII-85-8_x 0.40 0.045108021 0.681 CDBox 135 HBII-85-27_x 0.29 0.045678744 0.651 CDBox 56 miR-99b-star 0.49 0.049531821 0.651 miRNA 74

TABLE 8 50 miRNAs most significantly expressed between nevi and normal skin Log2- Probe Name FC P value AUC ProbeType #Sample Detected -14 −3.87  3.6592E−27 1.000 CDBox 56 14qll-14_x −3.21  3.815E−24 1.000 CDBox 56 U74_x 1.46 3.89068E−23 1.000 CDBox 135 miR-509-3p 5.78 1.67661E−22 0.997 miRNA 116 miR-768-5p −1.46 5.23441E−22 1.000 miRNA 135 ENSG00000199411_s 1.36 1.56796E−21 1.000 snoRNA 135 Z17B −1.94 2.89347E−20 0.997 CDBox 117 miR-149 −3.46 1.55425E−19 1.000 miRNA 100 miR-125a-5p −3.44 1.86526E−19 1.000 miRNA 128 U43 −1.19 3.32514E−19 1.000 CDBox 135 miR-1308 1.55  8.1184E−19 0.964 miRNA 135 U59A 1.13 9.99768E−18 0.993 CDBox 135 miR-146a 4.44 3.16232E−17 0.975 miRNA 129 miR-513a-5p 3.77 1.05679E−16 0.972 miRNA 105 ACA20 −1.74 1.56297E−16 0.993 HAcaBox 131 U43_x −1.05 1.04459E−15 1.000 CDBox 135 U44_x −1.37 5.54123E−15 0.994 CDBox 135 miR-127-3p −3.56 7.53549E−15 0.988 miRNA 89 miR-193b −2.57 7.81928E−15 1.000 miRNA 130 U83 −1.25 9.30493E−15 0.993 CDBox 135 miR-768-3p 0.98 2.39925E−14 0.972 miRNA 137 U33 −1.02 2.44194E−14 0.997 CDBox 137 miR-574-3p −3.05  3.0433E−14 0.974 miRNA 122 ACA16 −1.86 3.35128E−14 0.968 HAcaBox 47 14ql-4 −2.43 3.96317E−14 0.979 CDBox 80 hsa-let-7c −1.01 4.99111E−14 0.986 miRNA 135 miR-342-3p −2.06 1.67074E−13 0.996 miRNA 136 hsa-let-7b −1.01 2.25904E−13 0.964 miRNA 136 miR-423-3p −2.52 2.97967E−13 1.000 miRNA 107 HBII-239 −1.41  3.2162E−13 0.975 CDBox 135 U38B_x 1.52 3.80613E−13 0.968 CDBox 133 miR-320b −0.63 6.31909E−13 0.971 miRNA 135 miR-923 0.93 6.48332E−13 0.990 miRNA 135 U54 −1.06 1.17197E−12 0.975 CDBox 135 U38B 1.80 1.18885E−12 0.976 CDBox 134 U44 −1.07 1.28887E−12 0.981 CDBox 134 miR-139-5p −2.70 2.74261E−12 0.940 miRNA 80 miR-1826 −0.71 2.83317E−12 0.958 miRNA 137 ENSG00000199435 1.38 2.88249E−12 0.954 snoRNA 63 miR-26a 0.89 2.88328E−12 0.960 miRNA 136 U58B_x −1.47 3.60008E−12 0.940 CDBox 132 HBII-180A_x −1.43 4.25614E−12 0.992 CDBox 123 14qll-12_x −2.09 4.30741E−12 0.949 CDBox 85 miR-509-3-5p 3.46 5.53398E−12 0.953 miRNA 107 U55_x −1.35  6.7836E−12 0.996 CDBox 135 miR-149-star 0.93  9.6022E−12 0.940 miRNA 135 HBII-276 −1.41 1.19464E−11 0.964 CDBox 132 miR-23b −0.75 1.72105E−11 0.968 miRNA 137 miR-15b −2.42  1.7823E−11 0.968 miRNA 125 miR-921 1.73 1.93285E−11 0.954 miRNA 49

Example 4

The data from Example 3 was further analyzed to identify the combination of markers best able to distinguish (A) melanoma from normal skin and (B) melanoma from nevi. Each miRNA (“analyte”) was examined by svm, random forest, boosting, lasso, baggin, cart, matt, logistic regression, and ctree analyses. The use of multiple statistical method demonstrates that the best combination of markers identified by one statistical method is validated by every other method, making the choice of marker combinations less subject to the specific weaknesses of any one statistical algorithm.

FIGS. 1-10 show, respectively, the best 2-10 miRNA combinations for differentiating melanoma (MM) from normal skin (“NS”) and the relevant error rates, as determined by different statistical algorithms.

FIGS. 11-23 show, respectively, the best 2-14 miRNA combinations for differentiating melanoma (MM) from nevi (NV), and the relevant error rates, as determined by different statistical algorithms.

FIG. 24 shows the log 2 signal for three miRNA: miR-150 (R-150), miR-149-star (miR-149-star), and hsa-miR-1308 (miR-1308) across 137 samples. Samples 1-19 are normal skin (NS), 20-57 nevi (NV), 58-115 melanoma (MM) and 116-137 metastatic melanoma (Mets). As can be appreciated, miR-150, miR-149-star and miR-1308 distinguish normal skin and nevi from melanoma and metastatic melanoma.

Example 5

In a follow up study, a total of 78 melanoma and 98 nevi (Intradermal-20, Compound-18, Junctional-20, Blue-20, Spitz-20) were studied on microarray. The candidate microRNAs were chosen based on AUC, p value, log 2 fold change, and 9 analysis programs, as before. A example of error rate and AUC from 9 programs is set forth in FIG. 25. The candidate microRNAs identified from 2 programs (random forest and boosting) are listed in Table 9, below.

TABLE 9 MicroRNA candidates from 78 melanoma and 98 nevi microrna adj.P.Val (FDR) AUC Log2FC Order in program miR-1268 1.45E−19 0.89 −1.80 5 miR-1228-star 2.87E−19 0.91 −1.77 2 miR-92b-star 4.62E−18 0.88 −1.73 3 miR-155 5.56E−18 0.86 2.85 4 miR-345 1.19E−16 0.85 1.83 7 miR-425 5.11E−16 0.85 1.80 miR-132 5.24E−16 0.85 1.75 10 miR-1207-5p 1.18E−15 0.86 −1.40 miR-1301 2.17E−15 0.83 1.70 miR-663 2.26E−15 0.85 −1.43 miR-339-5p 2.26E−15 0.85 1.60 9 miR-149-star 5.28E−15 0.85 −1.32 miR-150 5.25E−14 0.82 2.10 6 miR-18a 5.80E−13 0.8 0.84 miR-103 1.52E−11 0.86 0.93 8 miR-191 7.32E−13 0.9 0.85 1 miR-296-3p 1.05E−09 0.79 −0.85 11 miR-31 1.39E−12 0.79 2.46 miR-107* 3.27E−10 0.85 0.86 miR-93* 1.05E−09 0.85 1.14 miR-1275* 4.59E−11 0.8 −1.16 miR-181B* 3.69E−12 0.83 1.69 miR-921* 7.21E−12 0.81 −0.89 miR-1225-5p 7.63E−10 0.78 −1.3 miR-1202 1.08E−09 0.76 −1.34 miR-342-3p 1.55E−10 0.78 1.29 Internal control candidates miR-27b 0.72 0.07 0.501 miR-195 0.95 0.01 0.506 miR-199b-3p 0.78 0.05 0.509 miR-199a-3p 0.86 0.03 0.511

REFERENCES

-   1. Leidinger P, Keller A, Borries A, Reichrath J, Rass K, Jager S U,     Lenhof H P, Meese E. High-throughput miRNA profiling of human     melanoma blood samples. BMC Cancer. 2010 Jun. 7; 10:262. -   2. Demetra Philippidou, Martina Schmitt, Dirk Moser, Christiane     Margue, Petr V Nazarov, Arnaud Muller, Laurent Vallar, Dorothee     Nashan, Iris Behrmann and Stephanie Kreis Signatures of MicroRNAs     and Selected MicroRNA Target Genes in Human Melanoma. Cancer Res     2010 May 70(10); 4163-4173 -   3. Segura F, Ilana Belitskaya-Lévy I, Rose A, Zakrzewski J,     Gaziel A. Melanoma MicroRNA Signature Predicts Post-Recurrence     Survival Clinical Cancer Research 2020 March 16(5); 1577-1586

The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

The inventions illustratively described herein may suitably be practiced in the absence of any clement or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including,” containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. 

What is claimed is:
 1. A method for differentially diagnosing between melanoma and nevus in a skin sample comprising: (a) measuring the level of two or more miRNAs selected from the group consisting of: (i) miR-132; (ii) miR-150; (iii) miR-339-5p; (iv) miR-15b; (v) miR-342-3p; (vi) miR-572; (vii) miR-155; (viii) miR-425; (ix) miR-1202; (x) miR-1268; (xi) HBII-382_s; (xii) miR-1225-5p; (xiii) miR-30c; (xiv) miR-106b-star; (xv) miR-125a-5p; (xvi) mgU6-53B; (xvii) miR-25; (xviii) miR-149-star; (xix) miR-939; (xx) miR-92b-star; (xxi) miR-500-star; (xxii) miR-22; (xxiii) HBII-142_x; (xxiv) miR-181b; (xxv) HBII-142; (xxvi) U38B; (xxvii) miR-663; (xxviii)miR-1224-5p; (xxix) miR-23a; (xxx) HBII-85-6_x; (xxxi) miR-1207-5p; (xxxii) miR-1301; (xxxiii) miR-1228-star; (xxxiv) miR-345; (xxxv) miR-30a-star; (xxxvi) ENSG00000199411_s; (xxxvii) ENSG00000202327; (xxxviii) miR-92a; (xxxix) miR-127-3p; (xl) HBII-85-26; (xli) miR-1308; (xlii) miR-31; (xliii) miR-921; (xliv) miR-146b-5p; (xlv) miR-768-3p; (xlvi) miR-708; (xlvii) miR-139-5p; (xlviii) ACA24_x; (xlix) miR-501-3p; (l) miR-502-3p; (li) miR-923, and (lii) miR-191; and (b) diagnosing the skin sample as containing melanoma when a difference in the level of the two or more miRNAs compared to a reference level indicates melanoma in the sample.
 2. The method of claim 1, comprising measuring the level of: (i) miR-150; and (ii) miR-149-star.
 3. The method of claim 1, comprising measuring the level of: (i) miR-150; (ii) miR-149-star; and miR-1308.
 4. The method of claim 1, comprising measuring the level of: (i) miR-150; (ii) miR-149-star; (iii) miR-1308; and (iv) miR-191.
 5. The method of claim 1, comprising measuring the level of: (i) miR-150; (ii) miR-149-star; (iii) miR-1308; (iv) miR-191; and (v) miR-1228-star.
 6. The method of claim 1, comprising measuring the level of: (i) miR-150; (ii) miR-149-star; (iii) miR-1308; (iv) miR-191; (v) miR-1228-star; and (vi) ENSG00000199411_s
 7. The method of claim 1, comprising measuring the level of: (i) miR-150; (ii) miR-149-star; (iii) miR-1308; (iv) miR-191; (v) miR-1228-star; (vi) ENSG00000199411_s; and (vii) miR-1268.
 8. The method of claim 1, comprising measuring the level of: (i) miR-150; (ii) miR-149-star; (iii) miR-1308; (iv) miR-191; (v) miR-1228-star; (vi) ENSG00000199411_s; (vii) miR-1268; and (viii) miR-923.
 9. The method of claim 1, comprising measuring the level of: (i) miR-150; (ii) miR-149-star; (iii) miR-1308; (iv) miR-191; (v) miR-1228-star; (vi) ENSG00000199411_s; (vii) miR-1268; (viii) miR-923; and (ix) miR-23a.
 10. The method of claim 1, comprising measuring the level of: (i) miR-150; (ii) miR-149-star; (iii) miR-1308; (iv) miR-191; (v) miR-1228-star; (vi) ENSG00000199411_s; (vii) miR-1268; (viii) miR-923; (ix) miR-23a; and (x) miR-132
 11. The method of claim 1, comprising measuring the level of: (i) miR-150; (ii) miR-149-star; (iii) miR-1308; (iv) miR-191; (v) miR-1228-star; (vi) ENSG00000199411_s; (vii) miR-1268; (viii) miR-923; (ix) miR-23a; (x) miR-132; and (xi) miR-1207.5p.
 11. The method of claim 1, comprising measuring the level of: (i) miR-150; (ii) miR-149-star; (iii) miR-1308; (iv) miR-191; (v) miR-1228-star; (vi) ENSG00000199411_(—s;) (vii) miR-1268; (viii) miR-923; (ix) miR-23a; (x) miR-132; (xi) miR-1207.5p; and (xii) miR-342.3p.
 12. The method of claim 1, comprising measuring the level of: (i) miR-150; (ii) miR-149-star; (iii) miR-1308; (iv) miR-191; (v) miR-1228-star; (vi) ENSG00000199411_s; (vii) miR-1268; (viii) miR-923; (ix) miR-23a; (x) miR-132; (xi) miR-1207.5p; (xii) miR-342.3p; and (xiii) U38B.
 12. The method of claim 1, comprising measuring the level of: (i) miR-150; (ii) miR-149-star; (iii) miR-1308; (iv) miR-191; (v) miR-1228-star; (vi) ENSG00000199411_s; (vii) miR-1268; (viii) miR-923; (ix) miR-23a; (x) miR-132; (xi) miR-1207.5p; (xii) miR-342.3p; (xiii) U38B; and (xiv) miR-155.
 13. The method of claim 1, wherein melanoma is diagnosed in the skin sample by alterations in the level of an miRNA compared to a reference level, with the alterations in at least two miRNA selected from the group consisting of: (i) miR-150 increase; (ii) miR-149-star decrease; (iii) miR-1308 decrease; (iv) miR-191 increase; (v) miR-228-star decrease; (vi) ENSG00000199411_s decrease; (vii) miR-1268 decrease; (viii) miR-923 decrease; (ix) miR-23a increase; (x) miR-132 increase; (xi) miR-1207.5p decrease; (xii) miR-342.3p increase; (xiii) U38B decrease; and (xiv) miR-155 increase.
 14. The method of claim 1, wherein the two or more miRNAs is selected from the group consisting of: (i) miR-1268, (ii) miR-1228-star, (iii) miR-92b-star, (iv) miR-155, (v) miR-345, (vi) miR-425, (vii) miR-132, (viii) miR-1207-5p, (ix) miR-1301, (x) miR-663, (xi) miR-339-5p, (xii) miR-149-star, (xiii) miR-150, (xiv) miR-18a, (xv) miR-103, (xvi) miR-191, (xvii) miR-296-3p, (xviii) miR-31. (xix) miR-107*, (xx) miR-93* (xxi) miR-1275*, (xxii) miR-181B*, (xxiii) miR-921*, (xxiv) miR-1225-5p, (xxv) miR-1202, and (xxvi) miR-342-3p.
 15. The method of claim 1, wherein melanoma is diagnosed in the skin sample by alterations in the level of an miRNA compared to a reference level, with the alterations in at least two miRNA selected from the group consisting of: (i) miR-1268 decrease, (ii) miR-1728-star decrease, (iii) miR-92b-star decrease, (iv) miR-155 increase, (v) miR-345 increase, (vi) miR-425 increase, (vii) miR-132 increase, (viii) miR-1207-5p decrease, (ix) miR-1301 increase. (x) miR-663 decrease, (xi) miR-339-5p increase, (xii) miR-149-star decrease, (xiii) miR-150 increase, (xiv) miR-18a increase, (xv) miR-103 increase, (xvi) miR-191 increase, (xvii) miR-296-3p decrease, (xviii) miR-31 increase, (xix) mi R-107* increase, (xx) miR-93* increase, (xxi) miR-1275* decrease, (xxii) miR-181B* increase, (xxiii) miR-921* decrease, (xxiv) miR-1225-5p increase, (xxv) miR-1202 decrease, and (xxvi) miR-342-3p increase.
 16. The method of claim 1, further comprising measuring the level of at least one miRNA as an internal control
 17. The method of claim 16, wherein the internal control is selected from the group consisting of: (i) miR-27b, (ii) miR-195, (iii) miR-199b-3p, and (iv) miR-199a-3p.
 18. The method of claim 1, further comprising isolating the nucleic acid from the skin sample.
 19. The method of claim 1, further comprising amplification of the nucleic acid from the skin sample.
 20. The method of claim 1, wherein the skin sample is formalin fixed paraffin-embedded.
 21. The method of claim 1, wherein said measuring comprises quantitative PCR.
 22. The method of claim 1, further comprising histopathological assessment.
 23. The method of claim 1, further comprising (a) scoring (i) clinical observations (ii) histopathology observations and (iii) miRNA levels; and (b) combining (i), (ii) and (iii) to obtain a diagnostic score, that reflects the likelihood of melanoma.
 24. A kit for differentially diagnosing between melanoma and nevus in a skin sample comprising primers for the amplification of at least two miRNA selected from the group consisting of: (i) miR-150; (ii) miR-149-star; (iii) miR-1308; (iv) miR-191; (v) miR-1228-star; (vi) ENSG00000199411_s; (vii) miR-1268; (viii) miR-923; (ix) miR-23a; (x) miR-132; (xi) miR-1207.5p; (xii) miR-342.3p; (xiii) U38B; and (xiv) miR-155.
 25. A kit for differentially diagnosing between melanoma and nevus in a skin sample comprising primers for the amplification of at least two miRNA selected from the group consisting of: (i) miR-1268, (ii) miR-1228-star, (iii) miR-92b-star, (iv) miR-155, (v) miR-345, (vi) miR-425, (vii) miR-132, (viii) miR-1207-5p, (ix) miR-1301, (x) miR-663, (xi) miR-339-5p, (xii) miR-149-star, (xiii) miR-150, (xiv) miR-18a, (xv) miR-103, (xvi) miR-191. (xvii) miR-296-3p, (xviii) miR-31, (xix) miR-107*, (xx) miR-93*, (xxi) miR-1275*, (xxii) miR-181B*, (xxiii) miR-921*, (xxiv) miR-1225-5p, (xxv) miR-1202, and (xxvi) miR-342-3p.
 26. The kit of claim 21, further comprising primers for detecting at least one internal control selected from the group consisting of: (i) miR-27b, (ii) miR-195, (iii) miR-199b-3p, and (iv) miR-199a-3p. 